Useful combinations of restriction enzymes

Abstract

Compositions and methods are disclosed in which more than one restriction enzyme, including methylation-sensitive and / or methylation-dependent restriction enzymes, is used to analyze cfDNA. Useful combinations can be inactivated by heating to 65 DEG C, ideally more than 15 minutes. Restrictive digestion of cfDNA may occur for 11 hours or less. The digestion may be followed by an amplification and / or sequencing step.

Description

[0001] All literature and online information cited herein are incorporated by reference in their entirety. Technical Field

[0002] The present invention is in the field of analysis of methylation of cytosine residues in DNA and is particularly useful for analysis of human cell-free DNA, such as that found in plasma. background

[0003] A variety of techniques are known for analyzing the methylation of cytosine residues in DNA. A common method involves bisulfite conversion, in which unmethylated cytosine is converted to uracil using bisulfite. The converted DNA is then analyzed, and a comparison of the bisulfite-treated DNA and the non-bisulfite-treated DNA reveals which cytosine residues are not converted to uracil (and are therefore methylated). A major disadvantage of this technique is that bisulfite conversion is chemically harsh, resulting in high levels of degradation of the source material, which is a problem when using small amounts of source DNA. Chemical conversion is also biased and inherently noisy.

[0004] Another technique uses a methylation-sensitive restriction enzyme (MSRE), and if the cytosine in the CpG site in the recognition sequence of the enzyme is methylated, its activity is blocked. Various MSRE-based techniques using a single enzyme or a combination are available. For example, the HELP assay uses a combination of HpaII and MspI. The recognition sequences of both enzymes are CCGG, but HpaII is methylation-sensitive. Therefore, a comparison of the digestion products of the two enzymes can reveal which CCGG sites are CpG methylated. Other MSRE-based assays using multiple enzymes are known, including methods using three or four (or more) different enzymes.

[0005] Methylation-dependent restriction enzymes (MDREs) can also be used, which digest their recognition sequence only when the cytosine is methylated, ie the opposite of MSRE-based assays.

[0006] These enzyme-based techniques have also been used to analyze the methylation of cell-free DNA (cfDNA), such as in the EpiCheck platform sold by Nucleix. It has also been reported that cfDNA is digested using a variety of enzymes. For example, a mixture of HhaI, HpaII and exonuclease I has been used to digest cfDNA, and a mixture of two or three of BstUI, HhaI and / or HpaII has been used to analyze fetal cfDNA in maternal blood. The method of using BstUI alone or in combination with HhaI, HpaII or HpaII+HinP1I to digest cfDNA is known, and the method using Bsh12361+HhaI, Bsh12361+HpaII+HinP1I, HinP1I+HhaI, HhaI+HpaII or HhaI+AccII and the method using HpaII+AciI+HpyCH4IV and HinP1I are also known. Also known are DNA digestion methods using a mixture of two or three of BstUI+HpaII, HpaII+CfoI or AccII, HpaII and HpyCH4IV, and methods using BstUI+MluI, BstUI+HpaII or NauI+MbUi. Various of these methods involve downstream PCR steps, so it is necessary to inactivate the MSRE before PCR so that the amplicon (which will be unmethylated) is not digested.

[0007] There remains a need for additional MSRE and / or MDRE combinations that can be used to digest cfDNA and that can provide various advantages over combinations already used, including in techniques that include downstream PCR. There is also a need for additional techniques that use restriction enzyme combinations to analyze CpG methylation and that can provide advantages over known methods. Overview

[0008] The present invention provides a method for digesting cfDNA using a combination of restriction enzymes comprising HinP1I and AciI, wherein the method comprises the following steps: (i) digesting cfDNA with a restriction enzyme; and (ii) inactivating the restriction enzyme by heating for more than 15 minutes. Preferably, the restriction enzyme is inactivated by heating for 20 minutes or longer. Unlike the 15-minute heat inactivation used in some known methods, this inactivation period can achieve complete inactivation to ensure that residual enzyme activity does not continue into downstream steps.

[0009] The present invention also provides a method for digesting cfDNA using a combination of restriction enzymes comprising HinP1I and AciI, wherein the method comprises the following steps: (i) digesting cfDNA with restriction enzymes for 11 hours or less; and (ii) completely inactivating the restriction enzymes by heating. 11 hours of digestion is more than adequate for the digestion of all cfDNA in a typical sample, and a long digestion time of 14 hours or 16 hours is unnecessary in some known methods.

[0010] Although 11 hours or less of digestion is sufficient to completely digest cfDNA in a typical sample obtained from blood collected in a typical collection tube containing an anticoagulant (e.g., a K2EDTA collection tube), it has been found that digestion is inhibited in certain types of collection tubes. In particular, this inhibition can be seen in collection tubes containing an anticoagulant and an agent that inhibits the release of genomic DNA from leukocytes into the plasma component of a blood sample, such as BCT from Streck (see below). Therefore, when blood has been stored in such a tube, it may be useful to increase the digestion time compared to blood stored in a K2EDTA collection tube. Therefore, the present invention also provides a method for digesting cfDNA using a combination of restriction enzymes comprising HinP1I and AciI, wherein the method comprises the following steps: (i) providing a blood sample contained in a collection tube, the collection tube comprising an anticoagulant and an agent for inhibiting the release of genomic DNA from leukocytes in the sample into the plasma component of the blood sample; (ii) preparing plasma from the blood sample; and (iii) digesting cfDNA with a restriction enzyme for at least 2 hours. The method may further include (iv) a step of inactivating the restriction enzyme by heating, as disclosed elsewhere herein. The use of this type of collection tube advantageously enables the blood sample to be stored and / or transported (e.g., at room temperature) after collection while inhibiting contamination of the cfDNA with genomic DNA that would otherwise be released from the leukocytes into the plasma during storage. In some known methods, the 1 hour digestion time used after collecting blood from pregnant mothers in such tubes is sometimes found to be too short to obtain consistent and reliable results with cfDNA, and step (iii) may involve more than 2 hours of digestion (e.g., for at least 4, 6, 8, 10, 12, 14 hours or longer, such as for about 16 hours) and ideally be long enough to provide substantially complete digestion of the cfDNA. Contrary to previous reports that certain cfDNA blood collection tubes are not suitable for downstream analysis of methylated sequences in cfDNA, it has now been discovered that blood samples stored in these tubes can indeed be subjected to cfDNA analysis as disclosed herein, particularly when digestion occurs for 2 hours or longer.

[0011] The present invention similarly provides a method for digesting cfDNA using a combination of restriction enzymes comprising HinP1I and AciI, wherein the method comprises a step of digesting cfDNA with restriction enzymes for at least 2 hours, wherein the cfDNA is derived from plasma prepared from a blood sample contained in a collection tube, the collection tube comprising an anticoagulant and an agent for inhibiting the release of genomic DNA from leukocytes in the sample into the plasma component of the blood sample. The method may further comprise a step of inactivating the restriction enzyme by heating, as disclosed elsewhere herein.

[0012] The present invention similarly provides a method for digesting cfDNA using a combination of restriction enzymes comprising HinP1I and AciI, wherein the method comprises the following steps: (i) preparing plasma from a blood sample contained in a collection tube, the collection tube comprising an anticoagulant and an agent that inhibits the release of genomic DNA from leukocytes in the sample into the plasma component of the blood sample; and (ii) digesting cfDNA with a restriction enzyme for at least 2 hours. The method may include one or both of the following: before (i), a step of receiving a collection tube; and / or after step (ii), inactivating the restriction enzyme by heating, as disclosed elsewhere herein.

[0013] The present invention also provides a method for analyzing cfDNA, wherein the method comprises the following steps: (i) digesting cfDNA using a combination of restriction enzymes comprising HinP1I and AciI; and (ii) sequencing the digested cfDNA. In particular, step (ii) may involve next generation sequencing, wherein the digested cfDNA is converted into a sequencing library, and then the library is subjected to a sequencing reaction.

[0014] The present invention similarly provides a method for analyzing cfDNA, wherein the method comprises the step of sequencing a digested cfDNA sample, wherein the sample has been previously digested using a combination of restriction enzymes comprising HinP1I and AciI.

[0015] The present invention also provides a method for analyzing cfDNA, wherein the method comprises the following steps: (i) digesting cfDNA for 11 hours or less using a combination of restriction enzymes comprising HinP1I and AciI; (ii) completely inactivating the restriction enzymes; and (iii) performing real-time PCR on the digested cfDNA. As described above, 11 hours of digestion is sufficient to digest a typical amount of cfDNA, particularly when a downstream real-time PCR analysis is subsequently performed.

[0016] The present invention similarly provides a method for analyzing cfDNA, wherein the method comprises the following steps: (i) digesting cfDNA for 11 hours or less using a combination of restriction enzymes comprising HinP1I and AciI; (ii) completely inactivating the restriction enzymes; and (iii) performing real-time PCR on the digested cfDNA. As described above, 11 hours of digestion is sufficient to digest a typical amount of cfDNA, particularly when subsequently subjected to downstream real-time PCR analysis.

[0017] The present invention also provides a method for analyzing data derived from digested cfDNA, wherein the data includes real-time PCR quantitative cycle data or sequence read data from high-throughput sequencing, and the cfDNA is digested by a method for digesting cfDNA as disclosed herein. The method can be used to provide the methylation status of one or more CpG sites of interest in cfDNA.

[0018] The present invention also provides a composition comprising more than one restriction enzyme, wherein the more than one restriction enzyme consists of MSRE and / or MDRE, and wherein (i) at least two different restriction enzymes in the more than one restriction enzyme have different recognition sequences, and (ii) the restriction enzymes can be completely inactivated by heating to 65° C. Compared with using a combination of enzymes having the same recognition sequence (e.g., HhaI and HinP1I used by Zhao et al., both of which recognize GCGC but have different cleavage sites therein), by recognizing different sequences, the number of CpG sites of the genome that can be analyzed is increased. Inactivation at 65° C. is milder (e.g., in terms of the generation of undesirable ssDNA) and easier (e.g., less energy intensive) than inactivation of a mixture including enzymes such as HpaII, AvaI, HaeII or MluI (according to suppliers of such enzymes, it needs to be heated to 80° C. for inactivation), and provides a clear advantage compared to a mixture including enzymes that are not easily heat inactivated (e.g., as reported for BstUI, PvuI and HhaI).

[0019] The composition may be based on MSRE without requiring MDRE, and thus the invention also provides a composition comprising more than one MSRE, wherein (i) at least two different MSREs in the more than one MSRE have different recognition sequences, and (ii) the more than one MSRE can be completely inactivated by heating to 65° C. The composition may be free of MDRE.

[0020] The present invention also provides a composition comprising HinP1I and AciI as the only two restriction enzymes in the composition. This enzyme pairing covers more than 99% of the CpG islands in the human genome, and is simpler to prepare and more accurate than the more complex mixtures sometimes used. Compositions comprising only two restriction enzymes are advantageous because it is easier to adjust digestion conditions (e.g., incubation temperature, reaction buffer, etc.) for a combination of two enzymes without impairing enzyme activity compared to a combination of three or more enzymes. For example, Bsh1236I, HpaII, and HinP1I used by Ellinger et al. require different optimal buffers for 100% activity. ThermoFisher recommends "Tango buffer" as its best choice for HpaII and HinP1I, and for Bsh1236I, it recommends "buffer R". As reported by Ellinger et al., the use of only Tango buffer to prepare a reaction mixture comprising this enzyme combination therefore impairs Bsh1236I activity.

[0021] Both HinP1I and AciI showed 100% activity at 37°C and in the same reaction buffer, i.e., rCutSmart TM Both enzymes were also inactivated completely by heating to 65°C using the same diluent (Diluent A; NEB).

[0022] Compositions comprising only two restriction enzymes are also advantageous in downstream library preparation methods, which involve the depletion of small DNA fragments, which are not subsequently sequenced. Before sequencing, small DNA fragments are usually exhausted to remove free (i.e., unconnected) sequencing adapters and / or adapter dimers, otherwise they will interfere with the efficiency and / or quality of DNA sequencing. When the starting cfDNA molecule is cut at more than one site, more DNA fragments are provided, some of which are very small and are removed (and therefore not sequenced) during this step. Increasing the number of restriction enzymes used for digestion increases the possibility of producing these small DNA fragments, resulting in library preparation bias and underestimation of unmethylated CpG sites. Compositions comprising only two restriction enzymes limit this bias.

[0023] The invention also provides a composition comprising HinP1I and AciI, wherein the ratio of HinP1I to AciI is at least 1.2:1 (in enzyme units). It has been found that using an excess of HinP1I gives better results than the 0.5:1 or 1:1 ratios previously used. Without wishing to be bound by theory, it is believed that the improvement can occur because AciI can cut the human genome more frequently than HinP1I, and because a single cut is sufficient to impair PCR amplification, less AciI activity is required to achieve the same damage.

[0024] In these various methods and compositions, it is preferred that: (a) the ratio of HinP1I to AciI is at least 2:1; (b) Mg is provided to the restriction enzyme; ++ ion source; (c) the restriction enzyme is used at a pH above 7, such as in the range of 7.5-8.5; (d) the cfDNA is human cfDNA, such as human plasma cfDNA; and / or (e) the amount of cfDNA subjected to digestion is between 10 ng-400 ng, such as between 10 ng-250 ng or between 10 ng-200 ng.

[0025] The present invention also provides additional methods and compositions that include or use these compositions and / or methods, as described in detail below. Details

[0026] Methylation The methods and compositions disclosed herein can be used to analyze DNA methylation, and in particular to analyze the presence or absence of 5-methyl modifications of cytosine in the context of CG dinucleotide sequences (commonly denoted "CpG" dinucleotides or "CpG sites") in the DNA of eukaryotic organisms. CpG sites are not randomly distributed throughout the genomes of eukaryotic organisms, and are often found in clusters called "CpG islands". These islands have been formally defined (Gardiner-Garden & Frommer (1987) J Mol Biol 196: 261-82) as regions at least 200 bp long, with 50% or more GC content, and in which the observed to expected CpG ratio is greater than 60% (i.e., where the number of CpG sites multiplied by the length of the sequence, divided by the number of Cs multiplied by the number of Gs, is greater than 0.6). In mammalian genomes, CpG islands are often found near the start site of genes, and about 70% of promoters near the transcription start site in the human genome contain CpG islands. Methylation of more than one CpG site within a promoter's CpG island is often associated with stable silencing of gene expression from that promoter.

[0027] The human genome sequence contains approximately 28 million CpG sites (per haploid genome), with approximately 30,000 CpG islands. In any particular nucleated cell, some CpG sites will be methylated, while others will not. Methylation patterns can differ between different cells and tissues within a subject, such that a particular CpG may be methylated in one cell or tissue, but unmethylated in a different cell or tissue within the same subject.

[0028] It is known that tumors can display different methylation patterns compared to non-tumor cells (or compared to other types of tumors). Some sites can become overmethylated (hypermethylated) in tumors, while others can become undermethylated (hypomethylated), and differences in these patterns have been used to aid tumor diagnosis.

[0029] Cell-free DNA The methods and compositions disclosed herein are particularly suitable for analyzing cell-free DNA (cfDNA), i.e., fragmented genomic DNA found in body fluids in animals rather than in intact cells. The origin of cfDNA is not fully understood, but it is generally believed that it is released from cells during processes such as apoptosis and necrosis. Compared to intact genomic DNA, cfDNA is highly fragmented (see, for example, Alcaide et al. (2020) Scientific Reports10, article 12564), and typically circulates as fragments between 120-220 bp in length, with a peak of approximately 168 bp (in humans).

[0030] cfDNA is present in many body fluids, including but not limited to blood and urine, and the methods and compositions disclosed herein can use any suitable cfDNA source, such as blood samples (such as venous blood) or urine samples.Ideally, cfDNA is separated from blood, and blood can be processed to produce plasma (i.e., the liquid remaining after the whole blood sample is subjected to a separation process to remove blood cells, usually involving centrifugation) or serum (i.e., plasma without coagulation factors such as fibrinogen (fibrinogen)). Therefore, the methods and compositions disclosed herein can be used as part of a so-called liquid biopsy test, and can be implemented using plasma or serum cfDNA. Therefore, the methods disclosed herein can include the step of purifying cfDNA from blood, plasma or serum samples to provide cfDNA for digestion and analysis. The method can also include obtaining a blood sample and preparing plasma or serum therefrom, thereby providing a source for downstream cfDNA purification.

[0031] Blood can be collected in tubes containing an anticoagulant and an agent that inhibits the release of genomic DNA from leukocytes in the sample into the plasma component of the blood sample. Such tubes are commercially available from Streck (La Vista, NE) as glass cfDNA "blood collection tubes" or "BCTs", for example as discussed by Diaz et al. (2016) PLoS One 11(11): e0166354, and they can stabilize cfDNA in blood for up to 14 days at 6-37°C (thus providing an advantage over typical K2EDTA collection tubes). Useful anticoagulants include, but are not limited to, EDTA, heparin, or citrate. Useful agents for inhibiting the release of genomic DNA from leukocytes include, but are not limited to, diazolidinyl urea, imidazolidinyl urea, dimethylol-5,5-dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, glycinate), 5-hydroxy-methoxymethyl-1-aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxypoly [methyleneoxy]methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane, quaternary adamantane and mixtures thereof. Other useful components may include: quenchers (e.g., lysine, ethylene diamine, arginine, urea, adenine, guanine, cytosine, thymine, spermidine, or any combination thereof) that inhibit the reaction of free aldehydes with DNA in the sample; aurintricarboxylic acid; metabolic inhibitors (e.g., glyceraldehyde and / or sodium fluoride) and / or nuclease inhibitors.For example, the tube may include imidazolidinylurea (or diazolidinylurea), EDTA and glycine. Further information on suitable collection tubes may be found in WO2013 / 123030 and US2010 / 0184069.

[0032] Other useful collection tubes are available, including but not limited to various plastic tubes: "Cell-free DNA collection tubes" from Roche, made of PET; "LBgard blood tubes" from Biomatrica, made of plastic and suitable for up to 8.5 ml of blood; and "PAXgene blood DNA tubes" from PreAnalytiX or Qiagen. These different tubes are discussed in more detail in Kerachian et al. (2021) Clinical Epigenetics 13, 193, Schmidt et al. (2017) Clinica Chimica Acta 269: 94-8, and Grölz et al. (2018) Current Pathobiology Reports 6: 275–86.

[0033] These various tubes can store up to 8.5 mL of blood, or sometimes up to 10 mL. Thus, a blood sample taken from a subject may typically have a volume between 5-10 mL.

[0034] A 10 mL blood sample typically produces between 10-500 ng of cfDNA, but can sometimes produce significantly higher amounts, such as up to about 10 μg, particularly in certain cancer patients. The methods disclosed herein can be performed on the amount of cfDNA contained in a 10 mL blood sample. The methods and compositions disclosed herein can typically use 10-400 ng of cfDNA, such as 10-250 ng or 10-200 ng.

[0035] Preferably, cfDNA derived from plasma is analyzed. Kits for purifying cfDNA from plasma (and other body fluids) are readily available, such as the MagMAX cfDNA Isolation Kit from ThermoFisher, the Maxwell RSCccfDNA Plasma Kit from Promega, the Apostle MiniMax High Efficiency Isolation Kit from Beckman Coulter, or the QIAamp or EZ1 products from Qiagen.

[0036] Therefore, the methods and compositions disclosed herein can utilize cfDNA extracted from a biological fluid sample (typically from a plasma or serum sample) of a subject. The method can start from cfDNA that has been prepared, or can include an upstream step for preparing cfDNA. Similarly, the method can include an upstream step for obtaining a plasma sample before the step of preparing cfDNA from a plasma sample.

[0037] Preferably, the cfDNA used in the methods and compositions disclosed herein is substantially free of single-stranded DNA (ssDNA), i.e., less than 7% of the cfDNA molecules (by number) are single-stranded, and preferably less than 5% or less than 1% (i.e., so that at least 99% of the cfDNA molecules are double-stranded). In some embodiments, the cfDNA contains less than 0.1% ssDNA, less than 0.01% ssDNA, or may not even contain ssDNA (i.e., free of ssDNA). Extracting cfDNA to obtain a cfDNA sample substantially free of ssDNA is described in, for example, WO2020 / 188561. It is ensured that low levels of ssDNA avoid potential inhibition of restrictive digestion, and are also useful after digestion because ssDNA can interfere with downstream steps such as connection and amplification. Commercial kits can be used to quantify single-stranded DNA in samples, such as Promega QuantiFluor™ kits.

[0038] In some embodiments, all extracted cfDNA is used in the methods disclosed herein. In other embodiments, cfDNA is divided into more than one fraction, and one or more fractions are not used in the methods disclosed herein, but can be used for other analytical methods, or reserved for control experiments or for other purposes.

[0039] In some embodiments, cfDNA is quantified prior to digestion (e.g., by weight, by concentration, etc.). In other embodiments, cfDNA is not quantified prior to digestion.

[0040] The cfDNA used with the methods and compositions disclosed herein can be obtained from any eukaryotic subject such as a mammal, and is ideally obtained from a human subject. In some embodiments, the human subject may be known or suspected of having a disease (e.g., cancer). In other embodiments, the human subject may be known to be healthy. In some embodiments, the subject is not a pregnant woman.

[0041] Restriction enzymes and digestion The methods and compositions disclosed herein use restriction enzymes that recognize specific sequences in double-stranded DNA and introduce double-strand breaks into the DNA. The enzyme has a recognition site containing a CpG sequence. Type II restriction enzymes, i.e., enzymes in which double-strand breaks are introduced into the recognition site, are particularly useful. Using more than one restriction enzyme allows for simultaneous digestion in parallel within a sample.

[0042] More specifically, methods and compositions disclosed herein use methylation-sensitive restriction enzymes and / or methylation-dependent restriction enzymes. MSRE only cleaves target DNA when the CpG in its recognition site is not methylated, and methylation inhibits cleavage. On the contrary, MDRE only cleaves target DNA when the CpG in its recognition site is methylated. MSRE and MDRE are easily available from well-known commercial suppliers, such as ThermoFisher, New England Biolabs, Promega, etc.

[0043] MSREs include but are not limited to: AatII, AccII, AciI, AclI, AfeI, AgeI, Aor13HI, Aor51HI, AscI, AsiSI, AvaI, BceAI, BmgBI, BsaAI, BsaHI , BsiEI, BsiWI, BsmBI, BspDI, BspT104I, BssHII, BstBI, BstUI, Cfr10I, ClaI, CpoI, DpnII, EagI, Eco52I, FauI, FseI, FspI, HaeII, HapII, HgaI, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NaeI, NarI, NgoMIV, NotI, NruI, NsbI, PaeR7I, PluTI, PmaCI, PmlI, Psp1406I, PvuI, RsrII, SacII, SalI, ScrFI, SfoI, SgrAI, SmaI, SnaBI, SrfI, TspMI, ZraI.

[0044] MDRE includes, but is not limited to, BspEI, BtgZI, FspEI, GlaI, LpnPI, McrBC, MspJI, XhoI, and XmaI.

[0045] Method and composition disclosed herein can include more than one restriction enzyme, wherein more than one restriction enzyme is made up of MSRE and / or MDRE.Therefore, more than one restriction enzyme can include only MSRE, only MDRE or a mixture of the two (such as one or more MSRE plus one or more MDRE).However, in general, it is preferred to use MSRE, without MDRE, and therefore more than one restriction enzyme includes two or more MSRE.MSRE is used to produce cfDNA wherein methylated CpG sites are complete, but unmethylated CpG sites are digested.Therefore, for any specific restriction site containing CpG in cfDNA samples, compared with the cfDNA samples containing a higher percentage of methylation at the site, a higher percentage of methylation at the site causes a lower degree of digestion.

[0046] Preferred more than one MSRE include both HinP1I and AciI. In some embodiments, in addition to HinP1I and AciI, one or more MSREs can also be used, but more preferably HinP1I and AciI are used as the only two restriction enzymes for digesting cfDNA. This enzyme pairing covers more than 99% of the CpG islands in the human genome. For this MSRE pairing, it is preferred to include HinP1I in excess (in enzyme units) relative to AciI, and ideally at least 1.2: 1 (i.e. at least 1.2 units of HinP1I per unit AciI), such as at least 1.5: 1, at least 1.75: 1, at least 2: 1, at least 3: 1, at least 4: 1 or at least 5: 1. The ratio between 2: 1 and 5: 1 is particularly useful for human cfDNA, and an excess of about 4.5 is preferred, such as between 4.4 and 4.6. Digestion can be performed at about 37 ° C until completion. As described herein, incubation for 2 hours at 37°C is typically sufficient for complete digestion of cfDNA samples using HinP1I and AciI, but longer digestions may be used, for example when digesting cfDNA obtained from blood that has been stored in a blood collection tube that contains an anticoagulant and an agent that inhibits the release of genomic DNA from leukocytes into the plasma component of the blood sample (see elsewhere herein).

[0047] In some embodiments, HhaI, AspLEI or CfoI is used as one of the restriction enzymes for digesting cfDNA. HhaI, AspLeI and CfoI are each MSRE, and each recognizes the same recognition sequence as HinP1I, but not necessarily cleaves at the same cleavage site within the recognition sequence. In some embodiments, SsiI is used as one of the restriction enzymes for digesting cfDNA. SsiI is a MSRE and recognizes the same recognition sequence as AciI, but not necessarily cleaves at the same cleavage site within the recognition sequence. In some embodiments, HinP1I and SsiI are used as the only restriction enzymes for digesting cfDNA. In some embodiments, AciI and any one of HhaI, AspLEI and CfoI are used as the only restriction enzymes for digesting cfDNA. Advantageously, AciI and HinP1I can be completely inactivated by heating to 65°C, while HhaI and ASPLEI are not sensitive to heat inactivation. Furthermore, while both AciI and HinP1I use the same optimal buffer to obtain 100% activity, the recommended optimal reaction buffer is different for each of SsiI, AspLEI, and CfoI, making it more difficult to customize digestion conditions for these alternative enzyme combinations without compromising enzyme activity.

[0048] In some embodiments, any combination of MSREs that recognize the same recognition sequence as Hinp1I or AciI, but do not necessarily cleave at the same cleavage site within the recognition sequence, can be used to digest cfDNA.

[0049] The concentration of the restriction enzyme can be selected according to the specific experiment being performed. Typically, HinP1I can be used at 10-450 units / μg cfDNA, and AciI can be used at 2.5-100 units / μg cfDNA, for example, at a ratio of 4.5 units HinP1I / unit AciI. In other embodiments, HinP1I can be used at 500-2500 units / μg cfDNA, and AciI can be used at 100-500 units / μg cfDNA, for example, at a ratio of 4.4-4.6 units (such as 4.5 units) HinP1I / unit AciI. In terms of solution concentration, HinP1I can be used at 35-45 units / mL, and AciI can be used at 5-15 units / mL, for example, at a ratio of 4.5 units HinP1I / unit AciI.

[0050] HinP1I recognizes the sequence GCGC and cleaves after the first G to leave a two-nucleotide 5' overhang (5'-G / CGC). It cuts well at 37°C and can be heat-inactivated by heating at 65°C for 20 minutes. For HinP1I, NEB recommends using its rCutSmart™ buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg / mL recombinant albumin, pH 7.9). One unit of HinP1I is defined as the amount of enzyme required to digest 1 μg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl.

[0051] Some commercial suppliers provide Hin6I enzyme instead of HinP1I. These two enzymes have substantially the same properties, i.e., they have the same recognition sequence, the same optimal digestion temperature, and they can both be inactivated at 65°C for 20 minutes. In addition, 1 unit of Hin6I is defined in the same manner as 1 unit of HinP1I. Therefore, the terms HinP1I and Hin6I are used interchangeably herein, and any enzyme combination disclosed as using HinP1I should be understood to also disclose the same combination using Hin6I, for example, the present invention provides a combination of AciI and Hin6I in the same manner as disclosed herein for AciI and HinP1I.

[0052] AciI recognizes the sequence CCGC and cleaves after the first C to leave a two-nucleotide 5' overhang (5'-C / CGC). It cuts well at 37°C and can be heat-inactivated by heating at 65°C for 20 minutes. For AciI, NEB recommends using its rCutSmart™ buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg / mL recombinant albumin, pH 7.9). One unit of AciI is defined as the amount of enzyme required to digest 1 μg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Its recognition site is non-palindromic.

[0053] Lambda DNA is a commonly used DNA substrate extracted from bacteriophage lambda (cI857ind 1 Sam 7) and is 48502 bp in length. It is typically stored in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and is widely available from commercial suppliers, such as NEB, catalog number N3011S.

[0054] Because HinP1I and AciI share essentially the same digestion and inactivation conditions, they form a useful pair for digesting DNA. In contrast, enzymes such as HpaII, AvaI, HaeII, and MluI require heating to 80°C for inactivation. BstUI and PvuI and HhaI are not susceptible to heat inactivation. BstUI cuts best at 60°C and only shows 10% of its full activity at 37°C. PvuI only shows 10% of its full activity in NEB's rCutSmart™ buffer.

[0055] After digestion, the restriction enzyme is preferably inactivated, particularly if a downstream amplification step, such as PCR, is to be used. Heat inactivation is particularly suitable, and both HinP1I and AciI can be inactivated by heating the composition at 65°C for at least 20 minutes, for example between 20 minutes and 60 minutes. Further details on inactivation are given below.

[0056] Other useful combinations of enzymes include or consist of: (i) HinP1I+AciI+McrBC; (ii) HinP1I+AciI+MspJI; (iii) HinP1I+AciI+HpaII+HpyCH4IV+BstUI; (iv) HinP1I+AciI+HpaII+HpyCH4IV+AvaI; (v) MspJI+FspEI; (vi) MspJI+HinP1I+AciI; (vii) MspJI+FspEI+HinP1I+AciI; or (viii) MspJI+FspEI+HinP1I+AciI+HpyCH4IV.

[0057] Other useful enzyme combinations include or consist of MSREs that recognize the same recognition sequence as HinP1I and / or AciI, but not necessarily cleave at the same cleavage site within the recognition sequence, including: (i) AciI+HhaI; (ii) AciI+AspLEI; (iii) AciI+CfoI; (iv) SsiI+HinP1; (v) SsiI+HhaI; (vi) SsiI+AspLEI; (vii) SsiI+CfoI; (viii) SsiI+HinP1I. MspJI shares essentially the same digestion and inactivation conditions as HinP1I and AciI (e.g., it is active at 37°C in rCutSmart™ and can be inactivated at 65°C). These three enzymes can provide 85% CpG coverage and 100% CpG island coverage, and are therefore particularly useful.

[0058] Two other useful combinations include or consist of: (i) HinP1I+AciI+HpaII; or (ii) HinP1I+AciI+HpaII+HpyCH4IV. For both combinations, the methods and compositions of the present invention should use at least one of the following additional features, as discussed elsewhere herein: (a) HinP1I is used in excess of AciI in terms of enzyme units; (b) digestion occurs for 11 hours or less; (c) digested cfDNA is sequenced.

[0059] Other useful enzyme combinations include or consist of an MSRE that recognizes a recognition sequence that includes a recognition sequence of HinP1I and / or AciI, including: (i) AciI and one of the following: AfeI, Aor51HI, AscI, BssHII, PauI, HaeII, Eco47III, EheI, FspAI, GIaI, KasI, MteI, NarI, NsbI, PluTI, SfoI, or SgsI; (ii) BsrBI and one of the following: AfeI, Aor51HI, AscI, BssHII, PauI, HaeII, Eco47III, EheI, FspAI, GIaI, KasI, MteI, NarI, NsbI, PluTI, SfoI, or SgsI; (iii) MbiI and one of the following: AfeI, Aor51HI, AscI, BssHII, PauI, HaeII, Eco47III, EheI, FspAI, GIaI, KasI, MteI, NarI, NsbI, PluTI, SfoI, or SgsI; (iv) NotI and one of the following: AfeI, Aor51HI, AscI, BssHII, PauI, HaeII, Eco47III, EheI, FspAI, GIaI, KasI, MteI, NarI, NsbI, PluTI, SfoI, or SgsI; (v) SacII and one of the following: AfeI, Aor51HI, AscI, BssHII, PauI, HaeII, Eco47III, EheI, FspAI, GIaI, KasI, MteI, NarI, NsbI, PluTI, SfoI, or SgsI; (vi) Cfr42I and one of the following: AfeI, Aor51HI, AscI, BssHII, PauI, HaeII, Eco47III, EheI, FspAI, GIaI, KasI, MteI, NarI, NsbI, PluTI, SfoI, or SgsI; (vii) SgrBI and one of the following: AfeI, Aor51HI, AscI, BssHII, PauI, HaeII, Eco47III, EheI, FspAI, GIaI, KasI, MteI, NarI, NsbI, PluTI, SfoI, or SgsI.

[0060] Other useful enzyme combinations include or consist of an MSRE that recognizes a recognition sequence that includes a recognition sequence of HinP1I and / or AciI, including: (i) HinP1I and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (ii) AfeI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (iii) Aor51HI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (iv) AscI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (v) BssHII and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (vi) PauI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (vii) HaeII and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (viii) Eco47III and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (ix) EheI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (x) FspAI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (xi) GIaI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I, or SgrBI; (xii) KasI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I or SgrBI; (xiii) Mtel and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I or SgrBI; (xv) Narl and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I or SgrBI; (xvi) NsbI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I or SgrBI; (xvii) PIutI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I or SgrBI; (xviii) SfoI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I or SgrBI;(ixx) SgsI and one of the following: BsrBI, MbiI, NotI, SacII, Cfr42I or SgrBI. ;

[0061] When methods are described herein as involving "digestion", the term (and "digesting", etc.) refers to mixing an active restriction enzyme with the DNA under conditions where digestion can occur. If there is no recognition site for the restriction enzyme in question (e.g., because it is an MSRE and all recognition sequences are fully methylated), the "digestion" step still occurs even though DNA cleavage does not occur.

[0062] method Disclosed herein are various methods for digesting cfDNA using combinations of restriction enzymes, such as combinations of MSREs.

[0063] The enzyme and cfDNA are usually incubated for a long enough time for substantially complete digestion to occur, i.e., further incubation does not result in any measurable increase in cfDNA cleavage. For typical samples, this can be achieved by incubating at 37°C for 2 hours, but if necessary, a longer digestion can be performed, such as 3 hours, 4 hours, or longer (e.g., overnight). In some embodiments, digestion is performed for 11 hours or less. Therefore, in some embodiments, digestion can be performed between 2-11 hours, such as between 2-10 hours, between 2-9 hours, between 2-8 hours, or between 2-4 hours. In other embodiments (e.g., in the case of using a collection tube, as discussed herein), digestion can be performed for a longer period of time, such as 12 hours or longer.

[0064] After digestion occurs, it is preferred to inactivate the restriction enzyme, especially if a downstream amplification step will be used. Both HinP1I and AciI can be inactivated by heating them to 65°C, for example by immersing the reaction mixture in a 65°C water bath. Digestion reaction mixtures with cfDNA tend to have low volumes, so that the temperature of the entire reaction mixture reaches 65°C very quickly, resulting in enzyme inactivation. In some embodiments, heating at this temperature occurs for more than 15 minutes, and ideally occurs for at least 20 minutes, such as 20-60 minutes. If necessary, the temperature can exceed 65°C, but this is not required. This heating step is sufficient to completely inactivate the restriction enzyme, even if the digestion activity of the enzyme on the cleavable target cfDNA molecules is no longer measurably detected under the digestion conditions adopted before heating.

[0065] The present invention also provides methods for analyzing cfDNA, comprising digesting cfDNA as discussed above, followed by downstream analysis steps, e.g., amplification steps (such as PCR, and in particular real-time PCR), ligation steps (such as ligation of sequencing adapters), DNA sequencing steps, etc. See further below.

[0066] The present invention also provides a method for evaluating the methylation status of one or more CpG sites in cfDNA, comprising digesting cfDNA as discussed above, followed by a downstream analysis step of quantifying the degree of digestion at the one or more CpG sites. The degree of digestion can be determined individually for each site or in aggregate.

[0067] The present invention also provides a method for diagnosing the presence or absence of cancer in a subject, comprising evaluating the methylation status of one or more CpG sites in cfDNA as discussed above, wherein overmethylation and / or undermethylation of one or more CpG sites are associated with cancer. In some embodiments, the method includes preparing a report in paper or electronic form based on the assessment of the presence or absence of cancer, and optionally communicating the report to the subject and / or the subject's healthcare provider.

[0068] The present invention also provides a method for treating or managing cancer in a subject, comprising diagnosing the presence of cancer as described above, and administering a suitable anti-cancer therapy to the subject. Treatment may include one or more of surgical resection, chemotherapy, radiotherapy, immunotherapy and / or targeted therapy.

[0069] Preferred methods do not include a bisulfite conversion step. Other preferred methods include a step in which no chemical changes are made to the nucleobases in the DNA, such as no bisulfite conversion, no TAPS conversion, etc. TAPS conversion refers to TET-assisted pyridine borane sequencing.

[0070] Preferred methods do not use restriction enzyme isoschizomers, where one enzyme recognizes both methylated and unmethylated forms of a restriction site and the other enzyme recognizes only one of these forms.

[0071] Preferred methods do not use a cocktail of restriction enzymes where at least one enzyme has a recognition sequence that includes CpG but is neither a MSRE nor a MDRE, ie, an enzyme that digests regardless of the methylation status of CpG.

[0072] Some methods do not include a step of heating the sample containing purified cfDNA prior to digestion. Other preferred methods do not include such a pre-digestion heating step, which includes heating the sample to above 40°C, above 50°C, above 60°C, above 70°C, or to 80°C. Other preferred methods do not include a pre-digestion heating step that includes heating the sample at ≥80°C for ≥20 minutes.

[0073] Composition Various compositions comprising more than one restriction enzyme (eg, more than one MSRE) are disclosed herein. They are typically aqueous compositions comprising the enzymes in soluble active form as well as other components such as salts, buffers, cofactors, and the like.

[0074] These compositions can include salts and / or buffers in aqueous solutions. For example, the composition can include 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / mL recombinant albumin, pH 7.9 (i.e., the composition of the commercially available rCutSmart™ buffer). Alternatively, the composition can include 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 100 μg / mL recombinant albumin, pH 7.9 (i.e., the composition of the commercially available NEBuffer™ r3.1 product). pH is measured at 25°C.

[0075] The composition can include cfDNA, particularly when used for digestion. As discussed above, in some compositions, HinP1I is present at 10-450 units / μg cfDNA, and AciI is present at 2.5-100 units / μg cfDNA, for example, at a ratio of 4.5 units HinP1I / unit AciI. In other embodiments, HinP1I can be used at 500-2500 units / μg cfDNA, and AciI can be used at 100-500 units / μg cfDNA, for example, at a ratio of 4.4-4.6 units (such as 4.5 units) HinP1I / unit AciI. In terms of solution concentration, HinP1I can be present at 35-45 units / mL, and AciI can be present at 5-15 units / mL cfDNA, for example, at a ratio of 4.5 units HinP1I / unit AciI.

[0076] Thus, a useful composition of the invention comprises HinP1I and AciI (e.g., with an excess of HinP1I, as described herein), potassium acetate, Tris-acetate, magnesium acetate, albumin, pH 7.8-8.0 (and optionally, cfDNA to be digested). For example, the composition may comprise 4-5 units of HinP1I, 0.5-1.5 units of AciI, 50 mM potassium acetate, 20 mMTris-acetate, 10 mM magnesium acetate, 100 μg / mL albumin, pH 7.9, and cfDNA.

[0077] The restriction enzymes in the composition are preferably present in an enzymatically active form, as this allows them to be used to digest cfDNA. However, after digestion, the composition may be heated (e.g., to 65°C) to inactivate the enzyme, so in some embodiments, the restriction enzyme is present in a heat-inactivated form.

[0078] In some embodiments, the composition may also include PCR reagents, such as appropriate buffer / salt components (in addition to the buffer / salts that persist after digestion, if necessary), a DNA polymerase (such as Taq polymerase), dNTPs, primers, probes, and the like.

[0079] In some embodiments, the composition may also include sequencing reagents, such as one or more of the following: sequencing adapters, DNA ligases (such as T4 ligase), Klenow fragment of DNA polymerase I, A-tailing enzymes (such as Taq polymerase), blunt-end polymerases (e.g., T4 DNA polymerase), kinases (such as T4 polynucleotide kinase), and the like.

[0080] In some embodiments, the compositions may also include control DNA, as discussed below.

[0081] As described above, when the composition includes HinP1I and AciI, then HinP1I is ideally present in excess (in enzyme units) relative to AciI, and ideally in an excess of at least 1.2: 1, such as at least 1.5: 1, at least 1.75: 1, at least 2: 1, at least 3: 1, at least 4: 1, or at least 5: 1. For example, when it is intended to analyze human cfDNA, a ratio of at least 2: 1 is generally useful, and a ratio of about 4.5: 1 has been found to be useful when digesting human cfDNA from plasma.

[0082] Preferred compositions do not include restriction enzyme isoschizomers, where one enzyme recognizes both methylated and unmethylated forms of a restriction site and the other enzyme recognizes only one of these forms.

[0083] Preferred compositions do not include mixtures of restriction enzymes where at least one enzyme has a recognition sequence that includes CpG but is neither a MSRE nor a MDRE, ie, an enzyme that digests regardless of the methylation status of CpG.

[0084] Downstream amplification After digestion, the method disclosed herein may include a step of amplifying (e.g., PCR) the digested cfDNA. Typically, the amplification will target one or (preferably) more loci of interest, such as loci containing CpG sites whose methylation states are known or expected to be associated with a specific biological state (e.g., a cancer of interest). Therefore, upstream and downstream primers flanking the CpG site of interest are used, and if the intervening CpG-containing sequence is not digested by a restriction enzyme, the sequence will be amplified. The resulting amplicon can then be detected, for example, using a labeled probe complementary to a subsequence within the amplicon of interest.

[0085] Thus, the method may include the step of adding PCR reagents after digestion, such as suitable buffer / salt components (if necessary, in addition to digestion residual buffer / salts), DNA polymerase (such as Taq polymerase), dNTPs, primers and (optionally) probes. Alternatively, one or more of these components may be present during digestion, for example a hot start PCR protocol may be used so that the PCR reagents are already present during the digestion step, but they do not become active until the reaction mixture is heated (e.g. during heat inactivation of a restriction enzyme).

[0086] Restriction digestion is often performed in the presence of high levels of Mg ++ PCR usually depends on Mg ++ , so standard PCR buffers include Mg ++ However, in this case, the addition of standard PCR buffer would result in excess Mg ++ , which can inhibit amplification efficiency. Therefore, added PCR reagents can include lower levels of Mg than is usually the case. ++ .

[0087] When PCR primers and probes are present during MSRE digestion, they should be designed so that their sequences do not include the recognition site of the MSRE being used.

[0088] The amplification and detection of amplicon can be carried out by conventional PCR using fluorescently labeled primers, and then the amplified product is carried out by capillary electrophoresis. In some embodiments, after amplification, the amplified product is separated by capillary electrophoresis, and quantitative fluorescent signal. An electropherogram that draws the change of the fluorescent signal that changes with size (bp) or the time of injection can be generated, wherein each peak in the electropherogram corresponds to the amplified product of a single locus. Peak height (for example, using "relative fluorescence unit", rFU provides) can represent the intensity of the signal from the locus of amplification. Computer software can be used to detect peaks, and calculate the fluorescence intensity (peak height) of a group of loci that its amplified product runs on a capillary electrophoresis machine, and calculate the ratio between the signal intensity subsequently.

[0089] Preferred PCR technique is real-time PCR (also referred to as qPCR), wherein amplification and detection of amplified product are performed simultaneously. Real-time PCR can be used together with non-specific detection or sequence-specific detection. Non-specific detection (e.g., using dsDNA in combination with dyes, such as SYBR Green) can be used in methods disclosed herein, but if it is desired to distinguish more than one different amplicon in the same reaction, it is undesirable. Therefore, more typically, sequence-specific detection is used, and methods and compositions can use labeled oligonucleotide probes (usually with fluorophores and fluorescence quenchers on the same probe, such as in TaqMan systems), which are complementary to the specific sequences in the nucleic acid amplicon of interest. Different probes for the amplicon derived from different target CpGs can be labeled with different fluorophores so that more than one different amplicon can be distinguished.

[0090] Therefore, real-time PCR can be realized by using the hydrolysis probe based on the reporter molecule and quencher molecule of combination.In such mensuration, the oligonucleotide probe has the fluorescent part (fluorophore) attached to its 5' end and the quencher attached to 3' end.During PCR amplification, polynucleotide probe and its target sequence on template selectively hybridize, and along with polymerase, template is copied, due to the 5'-nuclease activity of polymerase, polymerase also cracks polynucleotide probe.When polynucleotide probe is complete, the close proximity between quencher and fluorescent part usually causes low-level background fluorescence.When polynucleotide probe is cracked, quencher is uncoupled from fluorescent part, causes the increase of fluorescence intensity.Fluorescent signal is relevant to the amount of amplified product, that is, signal increases along with amplified product accumulation.

[0091] Suitable fluorophores include, but are not limited to, fluorescein, FAM, lissamine, phycoerythrin, rhodamine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX, JOE, HEX, NED, VIC, and ROX. Suitable fluorophore / quencher pairs are known in the art and include, but are not limited to, FAM-TAMRA, FAM-BHQ1, Yakima Yellow-BHQ1, ATTO550-BHQ2, and ROX-BHQ2.

[0092] Fluorescence can be monitored during each PCR cycle, providing an amplification curve showing the change in the fluorescent signal from the probe as a function of cycle number. In the context of real-time PCR, the following terminology is used:

[0093] "Quantitative cycle" ("Cq") refers to the number of cycles in which fluorescence increases above a threshold value, which is set automatically by the software or manually by the user. In some embodiments, the threshold value can be constant for each CpG locus of interest and can be pre-set before amplification and detection. In other embodiments, the threshold value for each CpG locus can be defined separately after the run based on the maximum fluorescence level of each locus detected during the amplification cycle.

[0094] "Threshold" refers to the fluorescence value used for Cq determination. In some embodiments, the threshold can be a value above the baseline fluorescence and / or above the background noise and within the exponential growth phase of the amplification curve.

[0095] "Baseline" refers to the initial cycles of PCR where there is little change in fluorescence.

[0096] Computer software is readily available to analyze amplification plots and determine baseline, threshold, and Cq.

[0097] When CpG sites are not digested and therefore amplified in subsequent PCR, relatively low Cq values ​​are observed because detectable amplification products accumulate after a relatively small number of amplification cycles. In contrast, if amplicons are present at lower levels (e.g., because some of the CpG loci of interest are digested), fewer amplicons are observed and Cq values ​​are higher.

[0098] Therefore, these results can indicate, for any given CpG site, the proportion of cfDNA molecules in the sample that are methylated / unmethylated at that CpG site. These numbers can be expressed as percentages, fractions, normalized values, etc.

[0099] The length of primer can vary, and this depends on specific assay format and specific needs.In some embodiments, primer can be at least 15 nucleotides long, such as between 15-25 nucleotides or 18-25 nucleotides long.Primer can be modified to be suitable for selected amplification system.

[0100] Primers may be designed to generate an amplicon between 60-150 bp in length (when the relevant CpG site is intact), for example between 70-140 bp in length.

[0101] The length of the oligonucleotide probe can vary. In some embodiments, the probe can include between 15-30 nucleotides, 20-30 nucleotides, or 25-30 nucleotides.

[0102] Oligonucleotide probes can be designed to bind to either strand of a double-stranded amplicon.Additional considerations include the melting temperature of the probe, which should preferably be comparable to that of the primer.

[0103] In case of analyzing more than one CpG site in parallel, more than one target is amplified simultaneously in the same reaction mixture (co-amplification) using different primer pairs for each CpG site of interest, and these different primers can be designed so that they can work at the same annealing temperature during amplification. Thus, primers with similar melting temperatures (Tm) can be designed, for example, at about 100 nm with respect to each other. + Within 3° C.-5° C. Similar considerations apply when more than one probe is used.

[0104] Computer software is readily available for routine design of primers and probes to meet the various requirements of any particular experiment.

[0105] Downstream sequencing After digestion, the methods disclosed herein may include a step of DNA sequencing, such as a step using next generation sequencing ("NGS") technology (also referred to as high throughput sequencing). NGS generally involves three basic steps: library preparation; sequencing; and data processing. Examples of NGS technology include synthetic sequencing and ligation sequencing (e.g., those adopted by Illumina Inc., Life Technologies Inc., PacBio, and Roche), nanopore sequencing methods, and electronic detection-based methods, such as Ion Torrent™ technology (Life Technologies Inc.). NGS can be performed using a variety of high throughput sequencing instruments and platforms, including but not limited to: Novaseq™, Nextseq™, and MiSeq™ (Illumina), 454 Sequencing (Roche), Ion Chef™ (ThermoFisher), SOLiD® (ThermoFisher), and Sequel II™ (Pacific Biosciences). Appropriate platform-designed sequencing adapters are used to prepare sequencing libraries and are readily available from the manufacturer of the platform.

[0106] Library preparation for major high-throughput sequencing platforms involves the connection of specific adapter oligonucleotides (also referred to as "sequencing adapters") to the DNA fragments to be sequenced. Sequencing adapters typically include platform-specific sequences for fragment recognition of a specific sequencer, such as sequences that enable the connected molecules to bind to the flow cells of the Illumina platform (e.g., P5 and P7 sequences). Each sequencing instrument provider typically sells a specific set of sequences for this purpose. Further details of library preparation are discussed below.

[0107] Sequencing adapters can include a site for binding to a universal PCR primer set. This allows more than one adapter-ligated DNA molecule to be amplified in parallel by PCR using a single set of primers.

[0108] Sequencing adapters can include sample indexes, which are sequences that enable more than one sample to be combined and then sequenced together (i.e., multiplexed) on the same instrument flow cell or chip. Each sample index (usually 6-10 nucleotides) is specific to a given sample and is used for de-multiplexing during downstream data analysis to assign a single sequence read to the correct sample. Sequencing adapters can contain single or dual sample indexes, depending on the number of libraries combined and the desired level of accuracy.

[0109] Sequencing adapters can include unique molecular identifiers (UMIs) to provide molecular tracking, error correction, and increased accuracy during sequencing. UMIs are short sequences, typically 5 to 20 bases in length, that uniquely identify the original molecule in a sample library. Since each nucleic acid in the starting material is tagged, providing a unique molecular barcode, bioinformatics software can filter out duplicate reads and PCR errors with a high level of accuracy and report unique reads, removing identified errors before final data analysis.

[0110] In some implementations, the sequencing adapters include both the sample barcode sequence and the UMI.

[0111] In some embodiments, sequencing adaptors allow for paired-end sequencing.

[0112] In some embodiments, the compositions and methods disclosed herein use a Y-shaped sequencing adapter, i.e., an adapter consisting of two single-stranded oligonucleotides annealed to provide a double-stranded stem and two single-stranded 'arms'. In other embodiments, the compositions and methods disclosed herein use a hairpin sequencing adapter, i.e., a single-stranded oligonucleotide whose 5' and 3' ends are annealed to provide a double-stranded stem. For both the Y-shaped adapter and the hairpin adapter, the double-stranded stem can include a short single-stranded overhang, such as a single A or T nucleotide. For both the Y-shaped adapter and the hairpin adapter, the double-stranded stem can be connected to the cfDNA fragment to prepare a sequencing library.

[0113] Thus, suitable sequencing adapters for use in the compositions and methods disclosed herein may be TruSeq™ or AmpliSeq™ or TruSight™ adapters (for use on the Illumina platform) or SMRTbell™ adapters (for use on the PacBio platform).

[0114] In the case of addition of sequencing adapters by ligation, this typically occurs at both ends of the DNA to be sequenced.

[0115] Restriction digestion can leave blunt ends, but usually produces single-stranded overhangs. The library preparation step can retain the overhangs (i.e., add complementary nucleotides) or remove them. Since the sequence of the terminal single-stranded overhangs after digestion can include useful information, it is preferred to add sequencing adapters in a manner that retains the overhangs, such as using enzymatic ligation, in which a ligase covalently connects the sequencing adapters to the DNA fragments, wherein the terminal sequences of the adapters are complementary to the terminal sequences obtained using restriction enzymes, or by using a polymerase to add complementary nucleotides and produce blunt-ended fragments.

[0116] In addition to removing or filling in single-stranded overhangs, end repair methods performed prior to adapter ligation can ensure that DNA molecules contain 5' phosphate groups and 3' hydroxyl groups.

[0117] For some libraries, the incorporation of non-templated deoxyadenosine 5'-monophosphate (dAMP) into the 3' ends of blunt-ended DNA fragments (a process known as dA tailing) was used in library preparation. The dA tail prevents the formation of concatemers during downstream ligation steps and enables ligation of DNA fragments with adapter oligonucleotides having complementary dT overhangs.

[0118] As mentioned above, restriction digestion is usually carried out in the presence of high levels of Mg. ++ Sequencing library preparation may also depend on Mg ++ , so standard library preparation buffers include Mg ++ However, in this case, the addition of standard library preparation buffer can result in excess Mg ++ , which can inhibit the efficiency of downstream steps. Therefore, added reagents can include lower levels of Mg than is typically used for library preparation. ++ .

[0119] As the use of lower levels of Mg ++ As an alternative, chelators can be added after digestion, which can eliminate the need to remove or dilute excess Mg in downstream amplification steps. ++ It has been found that the addition of chelators at the concentrations disclosed herein does not impair either such amplification steps or subsequent sequencing. Chelators can be added to provide an amplification reaction mixture comprising a chelator and divalent cations in a molar ratio of between 1:20 and 2:1. For example, the reaction mixture can include 8-20 mM Mg ++ For example, about 10 mM magnesium. For example, the amplification may be carried out in a culture medium containing between 3-4 mM chelating agent and 4 mM Mg ++ The chelating agent may comprise one or both of EDTA and EGTA.

[0120] After library preparation, the prepared DNA molecules can be sequenced to provide more than one "sequence read". These sequence reads are then subjected to data processing, such as removing sequences that do not meet the required quality standards, removing duplicates, correcting sequencing errors, mapping sequences to a reference genome, counting the number of sequence reads, etc. Computer software is readily used to perform these steps.

[0121] Any specific CpG site can be characterized by more than one sequence read, and the more than one sequence read can be derived from the same original cfDNA molecule and / or derived from the sequence read of different cfDNA molecules across the same CpG site. Sequencing is suitably performed so that in at least 100 sequence reads, for example, in at least 200, 300, 400, 500, 600, 700 or more sequence reads, the CpG site of interest is seen. The number of sequence reads across a specific genomic locus (such as a specific CpG site) is referred to as sequencing "depth" or "coverage". The average number of sequence reads across a specific genomic locus (such as a specific CpG site) is referred to as average sequencing "depth" or "coverage". The term "mean" refers to the arithmetic mean. Therefore, the average depth can be calculated by dividing the sum of the nucleotides in the sequence reads mapped to the human genome by the length of the (haploid) human genome.

[0122] Higher sequencing depth improves accuracy by allowing signal to be better distinguished from noise. This is because high-throughput DNA sequencing is prone to errors (approximately 0.1%-10% of all called bases are incorrect), so sequence reads mapped to a specific locus often contain (apparent) mutations compared to the reference sequence at that site and / or other reads mapped to that site.

[0123] High depth can be used to determine whether differences in sequence reads relative to a reference sequence reflect the underlying sequence of the sample DNA (signal) or are due to errors during sequencing (noise). High depth can therefore help draw meaningful conclusions from sequencing data, especially about the presence of rare signals, such as those arising from tumor DNA. Methylated cfDNA molecules from tumors can be approximately 50% of total cfDNA. < 1% is present in plasma. Bisulfite sequencing does not provide sufficient depth at enough genomic sites to reliably detect these rare signals. In contrast, the method of the present invention interrogates CpG sites throughout the human genome at a very high average depth, thus allowing the detection of these rare signals.

[0124] Sequence reads can be mapped to a reference genome, i.e. a previously identified genomic sequence, whether partial or complete, assembled as a representative example of a species or subject. A reference genome is typically a haploid, and typically does not represent the genome of a single individual of a species, but a mosaic of the genomes of several individuals. The reference genome for the inventive method is typically a human reference genome, such as a complete human genome, such as the human genome assembly available at the National Center for Biotechnology Information website or at the University of California, Santa Cruz, Genome Browser. An example of a reference genome suitable for human research is "hg18" genome assembly. As an alternative, the updated GRCh38 primary assembly (up to patch p13) can be used.

[0125] Mapping aligns sequence reads to a reference genome to identify the position of the reads within the reference genome. Aligned sequence reads are designated as "mapped". The alignment process is intended to maximize the likelihood of obtaining regions of sequence identity across each sequence in the alignment, allowing for mismatches, insertions / deletions, and / or shearing of some short fragments at both ends of the reads. The number of sequence reads mapped to a genomic locus is referred to as the "read count" or "copy number" of that genomic locus. It is not necessary to map all of the sequence reads obtained; in fact, it is not uncommon that a portion of the sequence reads obtained in any given experiment will not be mapped.

[0126] The term "genomic locus" refers to a specific location within a genome, and may include a single location (a single nucleotide at a defined position in the genome) or a segment of nucleotides that start and stop at a defined position in the genome. One or more specific locations may be identified by molecular location, i.e., by the numbering of the starting base pair and the ending base pair on the chromosome and the chromosome. The genomic locus of interest herein contains at least one CpG site.

[0127] When restriction digestion uses MSRE, sequence reads across a particular CpG site are derived from undigested molecules, i.e., molecules methylated at the CpG site (for complete digestion). The methylation level of the CpG site can be calculated by dividing its read count by the expected read count of the site (e.g., if it is fully methylated and therefore undigested, the expected read count). The expected read count can be determined using, for example: (i) read counts of control loci not cut by restriction endonucleases; (ii) average read counts of more than one such control loci; or (iii) read counts of the same CpG site in an undigested control sample, optionally corrected for sequencing depth differences.

[0128] Alternatively, the expected read count for a CpG site can be determined as the sum of the read count for that CpG site (indicating methylation) plus the sum of the read counts whose ends map to that CpG site (indicating unmethylation), taking into account any end repair that occurred during library preparation if necessary.

[0129] To avoid double counting, unmethylated CpG sites can be counted as sequencing reads with their 5' ends mapped to the site, as sequencing reads with their 3' ends mapped to the site, or as half of the sum of sequencing reads with their 5' ends or 3' ends mapped to the site. Because some library preparation methods can lead to depletion of small fragments that are then not sequenced (e.g., in CpG islands, where the starting cfDNA molecule is cleaved by MSREs at more than one unmethylated site, thereby providing 3 or more restriction fragments, some of which are very small), the observed number of unmethylated CpG sites may be lower than the true value in the original sample. This distortion can be resolved to some extent by using the larger of the number of reads with the 3' end mapped to the site and the number of reads with the 5' end mapped to the site (or using an average).

[0130] Thus, for any given CpG site, these calculations can provide the proportion of cfDNA molecules in a sample that are methylated at that CpG site. Conversely, similar calculations can provide the proportion of a particular CpG site that is unmethylated. These numbers can be expressed as percentages, fractions, normalized values, etc.

[0131] One way to express the coverage of a particular CpG site is called "Hitspan100," which refers to the number of sequence reads that span at least 50 nucleotides upstream and downstream of a particular CpG position. For example, a Hitspan100 of 90 at a particular CpG site means that there are 90 sequence reads that span the site, with at least 50 nucleotides upstream and downstream.

[0132] The methods disclosed herein do not require differential adapter tagging of methylated and unmethylated DNA molecules. The same adapter population can be used for all molecules.

[0133] Comparison The methods disclosed herein can utilize positive and negative controls. In some embodiments, one or more of the following can be analyzed in parallel:

[0134] ● A DNA control that does not contain the recognition sequence for the restriction enzyme used for digestion. If this DNA is digested, the method was not performed correctly.

[0135] ● A DNA control containing the fully methylated recognition sequence of the restriction enzyme used for digestion. If this DNA is digested when the method uses only MSRE, it indicates that the method was not performed correctly (the opposite is true for MDRE).

[0136] ● A DNA control containing the complete unmethylated recognition sequence of the restriction enzyme used for digestion. If this DNA is not completely digested when the method uses only MSRE, this indicates that the method was not performed correctly (and vice versa for MDRE).

[0137] These DNA controls can also be used as reference points for analysis, for checking the completeness of the digestion, etc. As mentioned above, for example, if fragments were obtained using MSRE digestion, it may be useful to know the expected read counts in a downstream NGS experiment, and one way to obtain this value is to look at the read counts for DNA that does not contain the MSRE recognition sequence, or to look at the read counts for DNA that contains the recognition sequence but is fully methylated.

[0138] For these purposes, it is preferred that the DNA control be similar in size and composition to the cfDNA molecule containing the CpG sites of interest. Thus, although synthetic DNA or PCR amplicons or bacterial plasmid DNA can be used as unmethylated controls, these are more useful if they are of a size similar to the cfDNA (e.g., long synthetic DNA, or appropriately sized restriction fragments prepared from a plasmid).

[0139] Control experiments can be performed inside or outside the sample. For internal controls, control DNA may already be present in the sample (e.g., cfDNA containing known universal (un)methylated CpG sites, or cfDNA without recognition sequences for the restriction enzymes used) and / or may be added (e.g., synthetic DNA added to cfDNA). Therefore, control DNA can be processed in combination with cfDNA and undergo the same conditions as cfDNA, so the method may involve co-amplification of restriction loci and control loci. For external controls, control DNA is subjected to the same treatment as cfDNA, but is not part of the same reaction mixture.

[0140] Therefore, control DNA, such as cfDNA, can be digested with restriction enzymes, and then downstream analysis steps, such as amplification, DNA sequencing, etc., are performed. Real-time PCR of a suitable control locus can give a result that can be used as a reference point. For example, the cfDNA at the CpG site of interest and the signal obtained from the control DNA (especially the control DNA that has never been digested by the restriction enzyme used) can be compared, and the signal ratio can be used to determine the degree of methylation at the CpG site of interest, because the signal ratio reflects the ratio of methylation. Therefore, it is not necessary to assess the absolute methylation level at the genomic locus, but the method disclosed herein is performed by calculating the signal ratio between the genomic locus analyzed and the control. This is in contrast to some conventional methylation analysis methods for distinguishing tumor-derived DNA from normal DNA, which require determining the actual methylation level at a specific genomic locus. Therefore, the method disclosed herein can eliminate the need for a standard curve and / or additional laborious steps involved in determining the absolute methylation level, thereby providing a simple and cost-effective procedure. When using an internal control, an additional advantage is obtaining signal ratios for loci amplified in the same reaction mixture under the same reaction conditions, which can help eliminate potential sources of error (e.g., potential differences between reaction mixtures, such as concentrations of template, enzyme, etc.).

[0141] Thus, the method using qPCR may involve calculating the signal intensity ratio between the co-amplified CpG sites after DNA digestion disclosed herein, thereby providing the methylation state of the CpG site. The methylation state can then be compared with a reference value (e.g., obtained from a healthy subject or from a subject with a known disease), and based on the comparison, a diagnosis can be derived. Thus, the method may include: co-amplifying CpG sites and control loci from restriction endonuclease digested DNA, thereby generating co-amplified products; determining the signal intensity of each generated co-amplified product; and calculating the ratio between the signal intensities of the co-amplified products of the CpG site and the control locus.

[0142] The quantification cycle (Cq) of each locus can be determined and 2(Cq对照基因座-Cq CpG位点) To calculate the ratio between the signal intensities of the co-amplified products, the ratio was calculated. In other words, the decrease in Cq relative to the control locus was determined and this value was used as an exponent of 2 to calculate the ratio.

[0143] Therefore, using qPCR or sequencing, based on the extent of digestion at any particular CpG site, a numerical value representing the degree of methylation of that CpG site in the cfDNA sample can be derived. This value can be expressed in a variety of ways, such as as a ratio or percentage of cfDNA molecules that are methylated at the CpG site, or as the intensity of the signal obtained from a particular CpG site, or as a ratio between the CpG site and a control locus, etc.

[0144] Systems and Kits The invention also provides various systems and kits.

[0145] The system may include a computer processor for executing and / or controlling the methods disclosed herein, and / or for processing the results, for example for performing calculations based on the results.A method at least partially implemented by a computer is provided.

[0146] The system or kit may include: a blood, plasma or serum sample of a human subject; a component for implementing the method disclosed herein to at least one CpG site; and computer software stored on a non-transitory computer-readable medium, the computer software being capable of instructing a computer processor to determine the methylation value of the at least one CpG locus based on the methylation assay. The software may also be capable of associating the methylation value with a diagnostic result or prediction, for example by comparing one or more methylation values ​​with one or more reference values ​​to assess the presence of a disease in a subject. The computer software may receive data from a qPCR and / or NGS experiment.

[0147] Components for practicing the methods disclosed herein encompass biochemical components (e.g., enzymes, primers, probes, NTPs, etc.), chemical components (e.g., buffers, reagents), and technical components (e.g., PCR systems, such as real-time PCR systems, and equipment such as tubes, vials, plates, pipettes).

[0148] The system may be capable of preparing a report based on the methylation values ​​and / or communicating the report to the subject and / or the subject's healthcare provider.

[0149] Computer software includes processor executable instructions stored on a non-transitory computer readable medium. Computer software may also include stored data. A computer readable medium is a tangible computer readable medium such as a compact disc (CD), magnetic storage, optical storage, random access memory (RAM), read-only memory (ROM), or any other tangible medium.

[0150] The computer-related methods and steps described herein are implemented using software stored on non-volatile or non-transitory computer-readable instructions that, when executed, configure or direct a computer processor or computer to perform the instructions.

[0151] Each of the systems, servers, computing devices, and computers described in this application can be implemented on one or more computer systems and configured to communicate over a network. They can also all be implemented on a single computer system. In one embodiment, the computer system includes a bus or other communication mechanism for communicating information and a hardware processor coupled to the bus for processing information.

[0152] The computer system also includes a main memory, such as a random access memory (RAM) or other dynamic storage device, coupled to the bus for storing information and instructions to be executed by the processor. The main memory may also be used to store temporary variables or other intermediate information during the execution of instructions by the processor. When such instructions are stored in a non-transitory storage medium accessible to the processor, the computer system is presented as a special-purpose machine that is customized to perform the operations specified in the instructions.

[0153] The computer system may include a read-only memory (ROM) or other static storage device coupled to the bus for storing static information and processor instructions. A storage device such as a magnetic disk or optical disk is provided and coupled to the bus for storing information and instructions.

[0154] The computer system may be coupled via the bus to a display for presenting information to a computer user.

[0155] Input devices including alphanumeric and other keys may be coupled to the bus for communicating information and command selections to the processor. Another type of user input device is a cursor control, such as a mouse, trackball, or cursor direction keys, for communicating direction information and command selections to the processor and for controlling cursor movement on a display.

[0156] The method disclosed herein can be performed by a computer system in response to a processor executing one or more sequences of one or more instructions contained in a main memory. Such instructions can be read into the main memory from another storage medium (such as a storage device). The execution of the instruction sequence contained in the main memory causes the processor to perform the processing steps described herein. In an optional embodiment, a hard-wired circuit can be used to replace or combine with software instructions.

[0157] Suitable storage media include any non-transitory medium that stores data and / or instructions that cause a machine to operate in a specific manner. Common forms of storage media include, for example, floppy disks, diskettes, hard disks, solid state drives, magnetic tape or any other magnetic data storage medium, CD-ROMs, any other optical data storage medium, any physical medium with a pattern of holes, RAM, PROM and EPROM, FLASH-EPROM, NVRAM, any other memory chip or cartridge.

[0158] Storage media is different from transmission media, but can be used in conjunction with transmission media. Transmission media participates in the transfer of information between storage media. For example, transmission media include coaxial cables, copper wires, and optical fibers, including the wires that make up the bus.

[0159] The present invention also provides a kit comprising: (i) a composition comprising more than one restriction enzyme as discussed above; and (ii) a component for analyzing cfDNA that has been digested with the composition. These components can be, for example, components for performing PCR or for preparing a sequencing library from digested cfDNA. For example, the kit can include one or more of the following: (a) a buffer solution, for example, containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / mL recombinant albumin, pH 7.9, or containing 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 100 μg / mL recombinant albumin, pH 7.9; (b) DNA polymerase, dNTP, primer and optionally one or more probes; (c) sequencing adapter; (d) enzyme solution, including DNA ligase and / or DNA polymerase; and / or (e) control DNA. Further details of these components (a) to (e) are discussed elsewhere herein.

[0160] The kit may include an instruction manual for practicing the methods as disclosed herein.

[0161] The kit may include a non-transitory computer-readable medium storing computer software including instructions that, when executed, configure or direct a computer processor to perform the method steps disclosed herein.

[0162] Disclaimers In some cases, the disclosure of WO2022 / 107145 (PCT / IL2021 / 051382) was excluded.

[0163] In some embodiments, the compositions and methods disclosed herein do not use a mixture of cfDNA from 50-60 people.

[0164] In some embodiments, the compositions and methods disclosed herein do not use between 760-810 ng of cfDNA (particularly, between 760-810 ng of cfDNA from healthy patients).

[0165] In some embodiments, the compositions and methods disclosed herein do not use 26 ng or 94 ng of cfDNA from untreated non-small cell lung cancer patients.

[0166] In some embodiments, the compositions and methods disclosed herein do not use 25-95 ng of cfDNA from untreated non-small cell lung cancer patients.

[0167] In some embodiments, the compositions and methods disclosed herein do not use the group consisting of 8 CpG sites located at positions chr1-11397653, chr17-17362652, chr17-71690026, chr3-121760779, chr12-49705230, chr1-8120128, chr2-39309230 and chr12-84283776 in the hg18 human genome assembly (as disclosed in Table 4 of PCT / IL2021 / 051382).

[0168] In some embodiments, the compositions and methods disclosed herein do not use a mixture of 10 units of HinP1I and 5 units of AciI.

[0169] In some embodiments, the compositions and methods disclosed herein do not use a 2:1 activity ratio of HinP1I:AciI.

[0170] summary The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, and molecular biology, which are within the skill of the art, and such techniques are fully explained in the literature. See, for example, Methods InEnzymology (Academic Press, Inc.), Green & Sambrook (2012) Molecular Cloning: ALaboratory Manual, 4th edition (Cold Spring Harbor Press), Ausubel et al., (eds) Short protocols in molecular biology, 5th edition (Current Protocols), Molecular Biology Techniques: An Intensive Laboratory Course, (Ream & Field, eds., 1998, Academic Press), Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology (Hodmann & Clokie, 2018), Basic Molecular Biology & Techniques - Recent Advances: Molecular Biology & Its Technique (Singh et al., 2021), etc.

[0171] The term "comprising" encompasses "including" as well as "consisting of," for example, a composition "comprising" X may consist of X alone, or may include additional things, such as X+Y.

[0172] The term "about" with respect to a value x is optional and means, for example, x ± 10%.

[0173] The word "substantially" does not exclude "completely", for example, a composition "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0174] The term "between" with respect to two values ​​includes both values, for example a range "between" 10 mg and 20 mg specifically encompasses 10, 15 and 20 mg.

[0175] Unless otherwise specified, a process comprising the step of mixing two or more components does not require any particular order of mixing. Thus, the components may be mixed in any order. Where there are three components, two components may be combined with each other, which combination may then be combined with a third component, and so on.

[0176] The various steps of a method may be performed at the same or different times, in the same or different geographic locations (eg, countries), and by the same or different people or entities. Example

[0177] The human genome sequence was analyzed for the presence of recognition sequences for various MSREs and MDREs. The proportions of the total CpG sites (about 28 million) accessible to various MSREs and MDREs in the genome are shown in Table 1, as follows:

[0178] Table 1 MSRE CpG coverage MDRE CpG coverage AciI / SsiI 14.56% JbXj 79.65% HpaII / HapII 8.14% Bq 56% HpyCH4IV / HpySE526I / MaeII / TaiI 7.63% FspEI 28% HinP1I / HhaI / CfoI / AspLEI 5.87% LpnPI 8.14% BstUI / AccII / Bsh1236I 4.97% AvaI 3.54% BsaA 2.46% BbI 0.95% AcII 0.58% SnaBI 0.45% BnB 0.37% ZlUT 0.25% PcqI 0.09% HaeII 3.91% IUsQ 0.15% PmII 1.97% When combinations of enzymes were used, CpG coverage increased, and these combinations could provide recognition sites in more than 99% of CpG islands in the human genome, as shown in Table 2: Table 2 combination CpG coverage CpG island coverage HpyCH4IV 8% 63% HinP1I / HhaI / CfoI / AspLEI 6% 94% HpaII / HapII / MspI 8% 95% AciI / SsiI 15% 98% AvaI 3.5% 79% BstUI / AccII / Bsh1236I 5% 91.84% HhaI+HpaII 14% 98% HhaI+BstUI 9% 96.94% HhaI+HinP1I 6% 94% HinP1I+AvaI 10% 96% HinP1I+HpaII 14% 98% HpaII+AciI 22% 99% HpyCH4IV+AciI 22% 99% HinP1I+AciI 20% 99.24% HpaII+CfoI 14% 98.51% HpaII+HpyCH4IV 16% 97.76% BstUI+MIuI 5% 91.84% BstUI+HpaII 13% 98.44% HpaII+PmII 9% 96.28% HhaI+Bsh12361 9% 96.94% AccII+HpaII 13% 98.44% AccII+HpyCH4IV* ≤13% HinP1I+AciI+HpaII 29% 99.57% HpaII+BstUI+HinP1I* ≤19% HinP1I+AciI+McrBC 59% 99.99% HinP1I+AciI+MspJI 85% 100% BstUI+HhaI+HpaII* ≤19% AccII+HpaII+HpyCH4IV* ≤21% Bsh12361+HpaII+HinP1I* ≤19% HinP1I+AciI+HpaII+HpyCH4IV 36% 99.74% HinP1I+AciI+HpaII+HpyCH4IV+BstUI 38% 99.77% HinP1I+AciI+HpaII+HpyCH4IV+AvaI 38% 99.78% HinP1I+HpaII+AciI+BstUI+HhaI* ≤34% HpaII+HinP1I+AciI+HpyCH4IV 36% 99.74% JbXj 80% 99.99% MspJI+FspEI 84% 100% MspJI+HinP1I+AciI 85% 100% MspJI+FspEI+HinP1I+AciI 88% 100% MspJI+FspEI+HinP1I+AciI+HpyCH4IV 89% 100% *The enzyme combination represented by (*) represents the enzyme combination in which the sum of the number of recognition sequences of each enzyme present in the human genome (hg18 genomic build) is calculated for CpG coverage. In the case where the recognition sequence of an enzyme overlaps with the recognition sequence of another enzyme and each recognition sequence comprises the same CpG site, the overestimation of CpG coverage is calculated. The CpG coverage of such enzyme combinations calculated represents the maximum CpG coverage (that is, if there is no overlap between the recognition sequences of the entire human genome). The maximum CpG coverage is represented by "≤" in the upper table 2, for example, "≤13%". For the enzyme combination not marked with (*), the overlap of recognition sequences is taken into account when calculating CpG coverage / CpG island coverage, so the related values ​​in the upper table 2 represent absolute CpG coverage / CpG island coverage values.

[0179] The methylation status of more than one site in a single CpG island tends to be the same (called co-methylation). Therefore, a single target site in a CpG island can be sufficient to obtain the actual situation of the methylation status of the entire island. Only the pairing of two enzymes HinP1I+AciI provides the coverage of CpG islands>99%, with the minimum complexity and rapid digestion in the reaction mixture. When other MSREs with the same recognition sequence are used (for example HhaI, AspLEI or CfoI replace HinP1I and / or SsiI replace AciI), the same high-level coverage is provided, but the preferred combination of AciI and HinP1I provides the following advantages: both enzymes can be completely inactivated by heating to 65°C, while HhaI and AspLEI are insensitive to heat inactivation. Furthermore, both AciI and HinP1I use the same optimal buffer to obtain 100% activity, whereas the optimal reaction buffer recommended by the supplier for each of SsiI, AspLEI, and CfoI is different, making it more difficult to customize digestion conditions for these alternative enzyme combinations without compromising enzyme activity.

[0180] HinP1I and AciI can be completely inactivated by heating to 65°C for 20 minutes, and the improved coverage from adding HpaII or HpaII+HpyCH4IV or HhaI+BstUI+HpaII (as in some known methods) does not justify the disadvantage from needing 80°C to inactivate HpaII. The same is true for adding BstUI, which cannot even be inactivated by heat.

[0181] HinP1I and AciI were used alone for human cfDNA digestion followed by qPCR for various loci. In order to obtain comparable ΔCq values ​​for each enzyme, it was necessary to use more units of HinP1I. This is probably because AciI cuts the human genome more frequently than HinP1I, and a single cut is sufficient to prevent PCR amplification. In a mixture of HinP1I+AciI, better results were found when an excess (in enzyme units) of HinP1I was used, with an excess between 4-fold and 6-fold being the most useful, and a 4.5-fold excess providing the best results (in terms of ΔCq for each different locus relative to the control locus).

[0182] The HinP1I+AciI pairing with excess HinP1I has been used to digest human plasma-derived cfDNA prepared and pooled from approximately 60 subjects. The purified cfDNA was mixed with the enzyme and incubated at 37°C for 2 hours, followed by inactivation at 65°C for 20 minutes. 2 hours is long enough to achieve complete digestion (unless blood is collected in a BCT from Streck, in which case longer times are usually required to achieve complete digestion).

[0183] A useful digestion mixture was prepared by mixing 11 μL rCutSmart™ buffer (10x concentration), 4.5 μL HinP1I (10,000 units / mL), 1 μL AciI (10,000 units / mL), and 93.5 μL cfDNA solution (containing intact cfDNA extracted from a single blood collection tube). Digestion at 37°C for 2 hours followed by heating at 65°C for 20 minutes provided good results.

[0184] The digested cfDNA was used to prepare sequencing libraries using the NEBNext Ultra DNA Library Prep Kit. Sequencing libraries were prepared by adding Illumina platform sequencing adapters using enzymatic ligation while retaining information at the ends of the DNA molecules. Whole-genome NGS was performed on the library using the Illumina NovaSeq 6000 sequencing platform with an S4 flow cell. Sequence reads for each sample were mapped against the complete human genome (hg18 genomic build). 9 Of the sequenced reads, 98.4% were mapped to the reference genome.

[0185] Any enzyme with a recognition sequence longer than any enzyme listed in Table 1 or Table 2 but comprising the recognition sequence will inherently have a lower genome-wide CpG coverage / CpG island coverage than the related enzyme. The larger the length of the recognition sequence, the less likely it is to occur in the genome, because the recognition sequence of a length greater than 4 nucleobases is statistically less likely to occur in the genome than the recognition sequence of a length of 4 nucleobases. Therefore, the combination of enzymes with a recognition sequence longer than the recognition sequence of AciI or HinP1I but comprising the recognition sequence of AciI or HinP1I can have high CpG coverage / CpG island coverage, but this will not be as high as the preferred combination of AciI and HinP1I. The list of MSRE / MDRE with a recognition sequence comprising an AciI recognition sequence (CCGC) or a HinP1I recognition sequence (GCGC) is provided in Table 3A and Table 3B below, respectively.

[0186] Table 3A - MSRE / MDRE with recognition sequences comprising the AciI recognition sequence (CCGC).

[0187] name Identification sequence BsrBI / MbiI CCGCTC NotI GCGGCCGC SacII / Cfr42I / SgrBI CCGCGG Table 3B - MSRE / MDRE with recognition sequences comprising the HinP1I recognition sequence (GCGC).

[0188] name Identification sequence AfeI / Aor51HI AGCGCT AscI GGCGCGCC BssHII / PauI GCGCGC HaeII RGCGCY Eco47III AGCGCT EheI GGCGCC FspAI TGCGCA GlaI <![CDATA[G m CG m C]]> KasI GCGCGC MteI <![CDATA[G m CG m CNG m CGC]]> NarI GGCGCC NsbI TGCGCA PluTI GGCGCC SfoI GGCGCC SgsI GGCGCGCC The CpG coverage and CpG island coverage of each enzyme listed in Table 3A and Table 3B were lower than those of AciI and HinP1I, respectively.

[0189] It will be understood that the foregoing describes the inventor's work by way of example only and that modifications may be made while remaining within the scope and spirit of the invention.

[0190] References WO 2005 / 090607 WO 2011 / 109529 WO 2014 / 078913 WO 2015 / 169947 WO 2020 / 188561 WO 2022 / 073012 WO 2022 / 107145 US 10,801,060 US 2020 / 0283840 Aleman et al . (2008) Br J Cancer ; 98(2), 466-473 Alhonen-Hongisto et al. (1987) Biochem J ; 242, 205-210 Bait et al. (2020) Genome ; 64(5):533-546 Bartlett et al. (1991) Somatic Cell & Molecular Genetics ; 17(1):35-47 Beikircher et al. (2018) Chapter 21 in DNA Methylation Protocols (ed.Jörg Tost), Methods in Molecular Biology, vol. 1708:407-424 Ellinger et al. (2009) J Urol 182:324-29 Ghosh & Sen-Mandi (2018) Tropical Plant Research ; 5(1):1-7 Gong et al. (2002) Cell ; 11(6);803-814 Khulan et al. (2006) Genome Res 16:1046-55 Larsson et al. (2012) Tree Genetics & Genomes ; 9:601-612 List et al. (1994) J. Biological Chemistry ; 269(16):11902-11911 Nalabothula et al. (2015) PLoS ONE 10(8):e0135410 Schmidt et al. (2017) Clinica Chimica Acta 469:94-8 From Roon et al. (2013) Clinical Epigenetics; 5(2):1-10 from Zogchel et al. (2021) JCO Precision Oncology 5:1738-1748 Wheelscher et al. (2015) EbioMedicine 2:929-36 Yan et al. (2009) Chapter 8 in DNA Methylation: Methods and Protocols (ed. Jörg Tost), Methods Molecular Biology; 507:89-106 Zhao et al. (2010) Prenat Diagn 30:778-82。

Claims

1. A method for digesting cfDNA using a combination of restriction enzymes consisting of HinP1I and AciI, wherein the method comprises the following steps: (i) digesting the cfDNA with the restriction enzyme; and (ii) inactivating the restriction enzyme by heating for more than 15 minutes, wherein the restriction enzyme is completely inactivated by the heating.

2. A method for digesting cfDNA using a combination of restriction enzymes consisting of HinP1I and AciI, wherein the method comprises the following steps: (i) providing a blood sample contained in a collection tube, the collection tube comprising an anticoagulant and an agent that inhibits the release of genomic DNA from leukocytes in the sample into the plasma component of the blood sample; (ii) preparing plasma from the blood sample; and (iii) digesting the plasma cfDNA with the restriction enzyme for at least 2 hours.

3. A method for analyzing cfDNA, wherein the method comprises the following steps: (i) digesting the cfDNA using a combination of restriction enzymes consisting of HinP1I and AciI to provide digested cfDNA; and (ii) sequencing the digested cfDNA.

4. A method for digesting cfDNA using a combination of restriction enzymes comprising HinP1I and AciI, wherein the method comprises the following steps: (i) digesting the cfDNA with the restriction enzyme for 11 hours or less; and (ii) inactivating the restriction enzyme by heating, wherein the restriction enzyme is completely inactivated by heating.

5. A method for analyzing cfDNA, wherein the method comprises the following steps: (i) digesting the cfDNA for 11 hours or less using a combination of restriction enzymes comprising HinP1I and AciI to provide digested cfDNA; (ii) completely inactivating the restriction enzymes; and (iii) performing real-time PCR on the digested cfDNA. The method according to claim 4 or claim 5, wherein the combination of restriction enzymes consists of HinP1I and AciI.

7. A composition comprising HinP1I and AciI as the only two restriction enzymes in the composition.

8. A composition comprising HinP1I and AciI, wherein the ratio of the activities of HinP1I to AciI is at least 1.2:

1.

9. A composition comprising more than one restriction enzyme, wherein the more than one restriction enzyme consists of MSRE and / or MDRE, wherein (i) at least two different restriction enzymes among the more than one restriction enzyme have different recognition sequences, and (ii) the restriction enzymes can be completely inactivated by heating to 65°C.

10. The composition of claim 9, comprising more than one MSRE, wherein (i) at least two different MSREs among the more than one MSREs have different recognition sequences, and (ii) the more than one MSREs can be completely inactivated by heating to 65°C.

11. The composition according to any one of claims 7-10, comprising at least one salt and / or at least one buffer; for example, comprising (i) 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / mL recombinant albumin, pH 7.9 or (ii) 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 100 μg / mL recombinant albumin, pH 7.

9.

12. The composition of any one of claims 7-11, comprising cfDNA.

13. The composition according to any one of claims 7 to 12, comprising PCR reagents and / or sequencing reagents.

14. A composition according to any one of claims 7 to 13, wherein: (a) the ratio of HinP1I to AciI is at least 2:1; (b) the composition comprises Mg ++ ions; and / or (c) the pH of the composition is above 7.

15. A method for analyzing cfDNA, comprising digesting the cfDNA according to any one of claims 1, 2 or 4, and then (a) performing real-time PCR on the digested cfDNA or (b) sequencing the digested cfDNA.

16. A method for evaluating the methylation status of one or more CpG sites in cfDNA, comprising digesting the cfDNA according to any one of claims 1, 2 or 4, and then quantifying the extent of digestion at one or more of the one or more CpG sites.

17. A method for diagnosing the presence or absence of cancer in a subject, comprising evaluating the methylation status of one or more CpG sites in cfDNA from the subject according to the method of claim 16, wherein overmethylation and / or undermethylation of the one or more CpG sites is associated with the cancer.

18. A method for treating or managing cancer in a subject, comprising diagnosing the presence of cancer in a subject according to the method of claim 17, and administering to the subject a suitable anti-cancer therapy.

19. The method according to any of the preceding method claims, wherein the restriction enzyme is inactivated after cfDNA digestion by heating the composition to 65°C for at least 20 minutes, wherein the restriction enzyme is completely inactivated by the heating.

20. The method according to any one of the preceding method claims, wherein: (a) the activity ratio of HinP1I to AciI is at least 2:1; (b) Mg is provided to the restriction enzyme during digestion ++ ion source; (c) digestion occurs at a pH greater than 7; (d) the cfDNA is human cfDNA; and / or (e) the amount of cfDNA subjected to digestion is between 10 ng and 400 ng.

21. The composition or method of any of the preceding claims, wherein the cfDNA is human plasma cfDNA.

22. A composition or method according to any preceding claim, wherein HinP1I is present in an excess (measured in enzyme units) of between 2:1 and 5:1 relative to AciI.

23. The composition or method of any preceding claim, wherein HinP1I is substituted by an MSRE that recognizes the same recognition sequence as HinP1I, and / or wherein AciI is substituted by an MSRE that recognizes the same recognition sequence as AciI.

24. A composition comprising (i) HinP1I, or an MSRE that recognizes the same recognition sequence as HinP1I, and (ii) AciI, or an MSRE that recognizes the same recognition sequence as AciI, as the only two restriction enzymes in the composition.

25. The composition of claim 24, wherein (i) the MSRE that recognizes the same recognition sequence as HinP1I is HhaI, AspLEI or CfoI, and / or (ii) the MSRE that recognizes the same recognition sequence as AciI is SsiI.

26. A composition comprising (i) any one of HhaI, AspLEI and CfoI and (ii) Ssil as the only two restriction enzymes in the composition.