Variant DNA quantification

Abstract

A method for quantifying a target DNA in a sample, the method comprising contacting (S1) the sample with N≥2 primer pairs each comprising a forward primer and a reverse primer, N labelled variant probes and a control probe. Each primer pair is designed to amplify a respective target portion of the target DNA, each labelled variant probe is complementary to or comprises a sequence variant of a respective target portion, the control probe is complementary to or comprises a wild-type sequence of the N target portions, the N labelled variant probes have a same first label. The method also comprises amplifying the N target portions by performing PCR amplification on the target DNA, detecting the first label bound to the N amplified target portions or released from the N labelled variant probes and quantifying the target DNA based on the detected first label.

Description

[0001] DNA QUANTIFICATION

[0002] TECHNICAL FIELD

[0003] It is a general objective to provide a quantification of target DNA molecules, and in particular a sensitive quantification that can quantify target DNA molecules present at low amounts in a sample.

[0004] BACKGROUND

[0005] There are several common techniques to quantify a target deoxyribonucleic acid (DNA) in sample including, for instance ultraviolet spectrophotometry, fluorometry, agarose gel electrophoresis and capillary electrophoresis. These techniques, however, require relatively high amounts of the target DNA in the sample in order to get a positive read-out. More sensitive methods are needed if the target DNA is expected to be present at low amounts. An example is circulating cell-free DNA (cfDNA) in a liquid biopsy sample. Analysis of such cfDNA can provide critical information about a patient’s health status, and include analysis of circulating skin-derived DNA, circulating tumor DNA (ctDNA), and circulating donor DNA. However, the amount of cfDNA in a liquid biopsy sample, such as blood sample, is miniscule and the target DNA is only a fraction, often less than 1 %, of the cfDNA. This extremely low abundance makes it very challenging to quantify the target DNA. As an example, extracting cfDNA from a 5 mL blood sample might result in a cfDNA sample where the number of copies of the target DNA is only a one-digit number. This leads to poor reproducibility with large uncertainty of the quantified amount and, in some cases, even a significant risk of missing the target DNA entirely. Theoretical limit of detection (LoD) at 95 % confidence is 3 molecules. Hence, with three target DNA molecules present in average in the extract from 5 mL blood, there is 5 % chance that an analysis is a false negative.

[0006] A common strategy to improve sensitivity is to increase the number of target DNA molecules in the sample. Real-time quantitative PCR (qPCR) is a highly sensitive method that quantifies target DNA by amplifying the target DNA and measuring the amount of DNA produced in real-time.

[0007] There is still a need for quantification of target DNA and in particular such quantifications that are sufficiently sensitive to quantify target DNA molecules expected to be present at very low amounts in a sample, such as cfDNA and ctDNA molecules.

[0008] SUMMARY

[0009] It is a general objective to provide a sensitive quantification of target DNA molecules in a sample.This and other objectives are met by embodiments of the present invention.

[0010] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0011] An aspect of the invention relates to a method for quantifying a target DNA in a sample. The method comprises contacting the sample with N primer pairs each comprising a forward primer and a reverse primer, N labelled variant probes and a control probe. N is an integer equal to or larger than 2 and each primer pair of the N primer pairs is designed to amplify a respective target portion ofthe target DNA. Each labelled variant probe of the N labelled variant probes is complementary to or comprises a sequence variant of a respective target portion of the N target portions. The control probe is complementary to or comprises a wild-type sequence of a target portion ofthe N target portions. The N labelled variant probes have a same first label. The method also comprises amplifying the N target portions by performing PCR amplification on the target DNA. The method further comprises detecting the first label bound to the N amplified target portions or released from the N labelled variant probes, and quantifying the target DNA based on the detected first label.

[0012] Another aspect ofthe invention relates to a method for quantifying a target DNA in a sample. The method comprises contacting the sample with N primer pairs each comprising a forward primer and a reverse primer, N variant padlock probes and a control padlock probe. N is an integer equal to or larger than 2, each primer pair of the N primer pairs is designed to amplify a respective target portion ofthe target DNA. Each variant padlock probe of the N variant padlock probes is complementary to or comprises a sequence variant of a respective target portion of the N target portions and the control padlock probe is complementary to or comprises a wild-type sequence of a target portion of the N target portions. The method also comprises joining 5’ and 3’ ends of padlock probes of the N variant padlock probes and the control padlock probe while hybridized to the target DNA to form circular variant padlock probes and circular control padlock probes. The method further comprises rolling circle amplifying the circular variant padlock probes with first labelled amplification primers having a first label and complementary to a portion of the circular variant padlock probes to generate first labelled rolling circle products and the circular control padlock probes with second labelled amplification primers having a second label different from the first label and complementary to a portion of the circular control padlock probes to generate second labelled rolling circle products. The method further comprises detecting the first labelled rolling circle products and the second labelled rolling circle products, and quantifying the target DNA based on the detected first and second labelled rolling circle products.The invention uses PCR assays for multiple target portions in a target DNA, but all PCR assays are designed to have the same readout by using a common label. Target specific primers are used in the PCR assays, but all assays use probes labelled with the same label. As a consequence, all these primers can then be added to a single PCR, where they will amplify multiple target potions in the target DNA that all generate the same labelled response. Hence, in a single PCR, the invention enables measurement of the total amount of all the target sequences. The invention thereby allows for a highly sensitive quantification of target DNAs but at a low cost.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

[0015] Fig. 1. dPCR assays designed for UV hotspot mutations distinguish mutant and wild-type sequences using artificial DNA gBIock fragments. UV-specific mutations arising at positions in the human genome that are hypersensitive to UV mutagenesis were detected with five assays targeted to the UV hotspots of CHCHD2 (chr7:56106490), CSPP1 (chr8:67062341), ERGIC3 (chr20:35542046), RPL15 (chr3:23917092) and RPL29 (chr3: 5199594) using FAM fluorophore for the mutant T and HEX fluorophore at the wild-type C. Artificial DNA fragments (g Blocks, 130-135 bp) containing the wild-type or mutant hotspot and surrounding sequence were diluted and 54,000 copies used to test the efficiency of the dPCR probes in 26,000 partitions on a QIAcuity dPCR instrument. 1D scatterplots showing the positive partitions for a given assay (contains both the wild-type HEX and mutant FAM probes for the indicated hotspots) in (A) 1-plex and (C) 5-plex assays, where all 5 assays were added in a single well. Line indicates positive partition detection threshold. The number of positive partitions is plotted for (B) 1-plex, and (D) 5-plex assays.

[0016] Fig. 2. Multiplexing of mutant assays shows increased sensitivity in UV-exposed DNA. 20 ng of DNA from A375 melanoma cells treated with 36 J / m2UVC for 6 weeks was diluted with increasing amounts of control untreated DNA to dilute the UV specific signal. UV specific mutations were detected with five UV specific assays targeted the promoters of CHCHD2 (chr7:56106490), CSPP1 (chr8:67062341), ERGIC3 (chr20:35542046), RPL15 (chr3:23917092) and RPL29 (chr3:5199594). (A) 1D scatterplot showing the positive partitions detected for a 5-plex of all assays, 3-plex (RPL15, ERGIC3, CSPP1 ) or individual assay (1-plex for RPL15). Horizontal line indicates the positive partition threshold. (B) The number of positive partitions is plotted for the 5-plex, 3-plex and 1-plex assays for each of the 4 dilutions (100 %, 33 %, 10%, 0 % UV). The 5-plex combination detects more positive partitions than the 3-plex and 1-plex assays only in the UV treated samples and decreases with decreasing UV DNA, whereas the wild-type assays remain the same.

[0017] Fig. 3 is a flow chart illustrating a method for quantifying a target DNA in a sample according to an embodiment.

[0018] Fig. 4 is a flow chart illustrating the contacting and amplifying steps of Fig. 3 according to an embodiment.

[0019] Fig. 5 is a flow chart illustrating an additional, optional step of the method shown in Fig. 3.

[0020] Fig. 6 is a flow chart illustrating a method for quantifying a target DNA in a sample according to an embodiment.

[0021] Fig. 7 schematically illustrates N assays running in a same PCR reaction according to an embodiment.

[0022] Fig. 8 schematically illustrates N assays running in a same PCR reaction according to another embodiment.

[0023] Fig. 9 schematically illustrates N assays running in a same PCR reaction according to a further embodiment.

[0024] Fig. 10 schematically illustrates one out of N assays running in a same PCR reaction according to an embodiment.

[0025] Fig. 11 schematically illustrates performing PCR pre-amplification according to an embodiment.

[0026] Fig. 12 schematically illustrates N assays running in a same PCR reaction according to an embodiment.

[0027] Fig. 13 schematically illustrates N assays running in a same PCR reaction according to an embodiment

[0028] Fig. 14. Double mutant probes targeting an ETS site in the RPL13A promoter provides increased sensitivity when applied to UV-exposed DNA compared to probes targeting single mutation, without increasing the number of probes. (A) 1 D scatterplot showing the positive partitions detected for two singlemutant probes and one double mutant probe. (B) The number of positive partitions is plotted for the two single mutant probes and one double mutant probe.

[0029] Fig. 15. Wildtype probes are replaced by competitor oligonucleotides containing 3' blocking C3 spacer groups. (A) 1D scatterplot showing the positive partitions detected for a first 5-plex assay (5-plex-1, CHCHD2, CSPP1, ERGIC3, RPL15, RPL29), (B) a second 5-plex assay (5-plex-2, RPL13A, SMUG1, PDCD11, ASXK2, DPH3), and (C) a 10-plex assay combining the two 5-plex assays. (D) The number of positive partitions is plotted for the two 5-plex assays and the 10-plex assay for each of the 4 dilutions (100%, 50%, 10%, 0% UV).

[0030] DETAILED DESCRIPTION

[0031] It is a general objective to provide a quantification of target DNA molecules, and in particular a sensitive quantification that can quantify target DNA molecules present at low amounts in a sample.

[0032] The present invention provides a quantification of DNA in a sample with improved sensitivity but where the quantification can be obtained with a “singleplex” assay or, when normalization is desired, a “duplex” assay. As a consequence of not requiring running multiple assays and then performing analysis based on multiplexing the levels of the different targets as quantified in each assay separated, the present invention can be performed at a shorter period of time and at substantially lower cost.

[0033] In brief, the invention uses assays for multiple target portions in a target DNA, but in contrast to using target specific readouts as in current practice, all assays are designed to have the same readout. Target specific primers are used in the assays, but all assays use probes labelled with the same label. As a consequence, all these primers can then be added to a single PCR, where they will amplify multiple target portions in the target DNA that all generate the same labelled response. Hence, in a single PCR, the invention enables measurement of the total amount of all the target sequences.

[0034] Fig. 3 is a flow chart illustrating a method for quantifying a target deoxyribonucleic acid (DNA) in a sample according to an embodiment. The method comprises contacting, in step S1 , the sample with N primer pairs, each comprising a forward primer and a reverse primer, N labelled variant probes and a control probe. According to the invention, the number N is an integer equal to or larger than 2. Each primer pair of the N primer pairs is designed to amplify a respective target portion of the target DNA. Further, each labelled variant probe of the N labelled variant probes is complementary to or comprises a sequence variant of a respective target portion of the N target portions and the control probe is complementary toor comprises a wild-type sequence of a target portion of the N target portions. The N labelled variant probes have a same first label.

[0035] A next step S2 comprises amplifying the N target portions by performing polymerase chain reaction (PCR) amplification of the target DNA. The method also comprises detecting the first label bound to the N amplified target portions or released from the N labelled variant probes in step S3 and quantifying the target DNA based on the detected first label in step S4.

[0036] The present invention thereby uses a plurality (N>2) of different primer pairs that are used to amplify a respective target portion, also referred to as target sequence, of the target DNA in the sample. Accordingly, each primer pair of the N primer pairs comprises a respective forward primer and a respective reverse primer. The amplification in step S2 thereby amplifies, if the target DNA is present in the sample, N target portions of the target DNA using the N primer pairs. The N amplified target portions can then be detected in the sample by means of the N labelled variant probes. At least one, preferably multiple and most preferably all, of the amplified target portions can either have a wild-type DNA sequence or a so-called sequence variant. An example of a sequence variant of a target portion is a mutation in the DNA sequence of the target portion, whereas the wild-type DNA sequence of the target portion is then the non-mutated DNA sequence. The N primer pairs are designed to amplify the N target portions regardless of whether the DNA sequence of a target portion is the sequence variant, such as mutated DNA sequence, or the wild-type sequence, such as a non-mutated DNA sequence. Each of the amplified target portions of the target DNA can thereby have the sequence variant, the wild-type sequence, or, the sequence variant and the wild-type sequence if the sample contained a corresponding target portion having the sequence variant and a corresponding target portion having the wild-type sequence.

[0037] Illustrative, but non-limiting, examples of sequence variants as used herein include mutated sequences containing one or more point mutations, insertions, deletions, inversions, translocations, etc. Sequence variants can also be variants of nucleotide modifications, such as base, sugar or backbone modifications.

[0038] The N labelled variant probes are each complementary to or comprises a sequence variant of a respective target portion of the N target portions. This means that the labelled variant probes will bind or hybridize to the corresponding amplified target portions if these amplified target portions have the respective sequence variant. Binding of a labelled variant probe to an amplified target portion will form a duplex between the labelled variant probe and the amplified target portion and where this duplex is labelled withthe first label. Each variant probe of the N labelled variant probes have the same first label and correspondingly each duplex between a variant probe and an amplified target portion will have the same first label.

[0039] In an embodiment, the N labelled variant probes are each complementary to a sequence variant of a respective target portion of the N target portions. In another embodiment, the N labelled variant probes comprises a sequence variant of a respective target portion of the N target portions. Generally, if the target DNA is double-stranded DNA it contains two hybridized strands. In such a case, the N labelled variant probes can target any of these strains. For instance and as further described herein, an example of a sequence variant is an E26 transformation specific (ETS) transcription factor binding site comprising an ETS hotspot mutation. Such an ETS hotspot mutation typically involves the mutation of a cytidine (C) into a thymidine (T) in connection with a core motif. In such an example, one of the strands in the wildtype sequence comprises a cytidine in connection with the core motif and the other strand comprises a guanine (G). One of the strands in the sequence variant then instead comprises a thymidine in connection with the core motif and the other strand comprises an adenine (A). The labelled variant probe could then target the strand containing the thymidine mutation by having a sequence complementary to the sequence variant with the thymidine mutation. Alternatively, the labelled variant probe could target the opposite strand by having a sequence comprising the sequence variant with the thymidine mutation. The control probe is complementary to or comprises a wild-type sequence of a target portion of the N target portions. This means that the control probe will bind or hybridize to the corresponding amplified portion having the wild-type sequence.

[0040] In an embodiment, the control probe is complementary to a wild-type sequence of a target portion of the N target portions. In another embodiment, the control probe comprises the wild-type sequence of a target portion of the N target portions.

[0041] I n a preferred embodiment, the control probe is complementary to a wild-type sequence of a target portion of the N target portions if the N labelled variant probes are each complementary to a sequence variant of a respective target portion of the N target portions.

[0042] In another preferred embodiment, the control probe comprises a wild-type sequence of a target portion of the N target portions if the N labelled variant probes comprises a sequence variant of a respective target portion of the N target portions.In an embodiment, the N labelled variant probes and preferably the control probe hybridize to the same strand, such as the sense strand or the antisense stand, of the target DNA. Such an embodiment may be beneficial to suppress cross-hybridization between the probes, in particular when the sequence variants and / or wild-type sequences have similar nucleotide sequences.

[0043] The control probe could be a non-labelled control probe, also referred to as unlabeled control probe, blocker or competitor oligonucleotide herein, or labelled control probe.

[0044] In the latter case, the labelled control probe has a second label different from the first label. In such an embodiment, step S3 comprises detecting the first and second labels bound to the N amplified target portions or released from the N labelled variant probes the labelled control probe. Step S4 comprises, in this embodiment, quantifying the target DNA based on the detected first and second labels.

[0045] Step S2 will thereby generate amplified target portions, to which labelled variant probes or labelled control probes are hybridized to form labelled duplexes. The first and second labels bound to the N amplified target portions are then detected and used to quantify the target DNA in steps S3 and S4. Alternatively, the first and second labels released from the N labelled variant probes or the labelled control probe are detected in step S3 and used to quantify the target DNA in step S4.

[0046] In the former case, the labels bound to the amplified target portions are detected in step S3. In the latter case, the labelled variant probes or labelled control probes are so-called hydrolysis probes, each comprising a reporter, also referred to as reporter dye, and quencher. In such a case, the quencher of a labelled variant probe or a labelled control probe suppresses or quenches any fluorescence emitted by the reporter of the labelled variant probe or the labelled control probe when the hydrolysis probe is intact via fluorescence resonance energy transfer (FRET). During the amplification in step S2, the hydrolysis probe binds to its complementary sequence (sequence variant or wild-type sequence). As the polymerase enzyme, such as Taq® polymerase, extends the DNA strand from the primer pairs, the polymerase enzyme encounters the hydrolysis probe and uses its 5’ to 3’ exonuclease activity to degrade the hydrolysis probe. As the hydrolysis probe is degraded, the reporter is released from the quencher, resulting in an increase in fluorescence, which can be measured in real-time.

[0047] Hence, in an embodiment, each labelled variant probe of the N labelled variant probes comprises a first reporter and a first quencher. The labelled control probe comprises a second reporter different from the first reporter and a second quencher or the first quencher. In this embodiment, the labelled variant probes and the labelled control probe are hydrolysis probes and could thereby be referred to as N labelled varianthydrolysis probes and a labelled control hydrolysis probe. In such a case, each labelled variant hydrolysis probe has the same first reporter (or first reporter dye) and the same first quencher, whereas the labelled control hydrolysis probe instead has a second reporter (or second reporter dye) that is different from the first reporter. The labelled control hydrolysis probe also comprises a quencher, which could be the same quencher as the labelled variant hydrolysis probes, i.e., the first quencher, or another quencher, i.e., the second quencher.

[0048] The present invention thereby runs N assays using N primer pairs in the same sample to amplify N target portions of a target DNA in the same reaction vessel. The N amplified target portions are then detected using the same first label through the N labelled variant probes. The present invention thereby quantifies the total number of variant sequences present in the target portions of the target DNA in the sample rather than identifying which variant sequences that were present in the target DNA in the sample. This enables usage of the same label for all N labelled variant probes and thereby for all target portions in the DNA, whereas the labelled control probe uses a different label than the N labelled variant probes. Such an approach is schematically illustrated in Fig. 7. The figure illustrates N amplified target portions of the target DNA in the sample. Each amplified target portion is detectable using a respective labelled variant probe each having the same label represented by a filled circle in the figure. In the example shown, the labelled control probe is designed to hybridize to a wild-type (WT) sequence of the first amplified target portion. The label of this labelled control probe, represented by filled square in the figure, is different from the labels of the N labelled variant probes. The amount of first label (filled circle) and second label (filled square) bound to the amplified target portions is then detected and used to quantify the target DNA present in the sample. In Fig. 7, “*” indicates a nucleotide in the first target portion that is mutated in the variant sequence as compared to the WT sequence.

[0049] Fig. 8 illustrates the corresponding approach using hydrolysis probes. The figure illustrates N amplified target portions of the target DNA in the sample. Each amplified target portion is detectable using a respective labelled variant hydrolysis probe each having the same reporter represented by a filled circle in the figure and the same quencher represented by a filled triangle in the figure. In the shown example, the labelled control hydrolysis probe is designed to hybridize to a wild-type (WT) sequence of the first amplified target portion. The reporter of this labelled control hydrolysis probe, represented by filled square in the figure, is different from the reporters of the N labelled variant probes. The labelled control hydrolysis probe also comprises a quencher represented by a filled diamond in the figure. The amount of fluorescence emitted from the first reporter and the amount of fluorescence emitted from the second reporter, obtained when the labelled variant hydrolysis probes and the labelled control hydrolysis probehave been degraded by the polymerase enzyme during the amplification, are then detected and used to quantify the target DNA present in the sample.

[0050] Fig. 9 illustrates an embodiment with multiple labelled variant probes with a same first label for multiple amplified target portions harboring variant sequences (N) and multiple labelled control probes with a same second label for multiple amplified target portions harboring wild-type sequences (M). In an embodiment, M = N. However, the invention is not limited thereto, i.e., in some applications M may be different than N.

[0051] Fig. 10 illustrates an alternative embodiment for the first target (target 1) in Fig. 7. In this embodiment, only a fraction of the control probes for the first target are labelled control probes and a remaining fraction of the control probes for the first target are non-labelled control probes. Such an embodiment could be advantages when there is a need to reduce the signal from the wild-type sequence relative to the signal from the variant sequence. Generally, the fraction of labelled control probes could be any percentage above 0 % but below 100 % in this embodiment. The fraction can vary for the different control probes.

[0052] In the examples illustrated in Figs. 7 and 8, the sample is contacted, in step S1 , with forward primer 1, reverse primer 1 , labelled variant probe 1 , control probe 1 , forward primer 2, reverse primer 2, labelled variant probe 2, ..., forward primer N, reverse primer N, and labelled variant probe N. The quantification of the target DNA is a multiplex, i.e., using N primer pairs, PCR amplification but using two detection channels, i.e., first label and second label. In this example, the labeled control probe is only present in one of the assays, i.e. for the first target portion. The examples as shown in Figs. 7 and 8 are particularly useful when target portions are present in 1:1 ratio, such as when quantifying genomic DNA. In such a case, the second label channel measures the amount of (genomic) target, while the first label channel measures a combined signal from all sequence variants of the target portions. Such an approach is cheaper and more sensitive as compared to prior art approaches that are based on a duplex reaction (variant sequence and wild-type sequence) for each target portion of the DNA target.

[0053] In an embodiment, multiple (M>2) assays are used for wild-type sequences too as schematically shown in Fig. 9. In such a case, all M assays having labelled control probes with the same second label. Hence, N assays are used for variant sequences all having one and the same first label and M assays are used for wild-type sequences all having one and the same second label. The embodiment is therefore analyzed as a duplex assay, with two signals: one for wild-type targets and one for variant targets. This embodiment is particular interesting when analyzing samples with unstable genomes, such as cancer cells, wheredeletions and copy number variations are common, and some wild-type and / or variant target sequences may be missing or be present in unnormal number of copies.

[0054] In another embodiment, multiple (N>2) assays are used to target variant sequences and multiple (M>2) assays are used to target wild-type sequences, but while essentially all assays for variant targets are using labeled variant probes only a fraction of the control probes of each assay for the wild-type sequence have labels, whereas a remaining fraction of the control probes do not contain any labels as shown in Fig. 10. The fraction of each control probe for a wild-type target that is labelled is preferably of the order of the expected variant sequence frequency. Hence, if variant sequence is expected to be present in about 1 % of the target DNA, then about 1 % of the wild-type probes are labelled. The objective is to balance the signals from the label of variant assays and the label of wild-type assays. In case labeled and unlabeled control probes for a target wild-type sequence are different, for example, having different lengths or chemical modifications, that affect their affinity for target DNA, this should be considered when choosing the ratio of labeled and unlabeled control probe.

[0055] An embodiment can be used to detect and preferably quantify the amount of a minor fraction of nucleic acid against a dominant fraction. This is, for instance relevant using liquid biopsy sampling to monitor, for example, patients that have undergone organ transplantation, in which case the amount of donor DNA relative to the recipient DNA is measured; pregnant women, in which case amount of fetal DNA relative to maternal DNA is measured; cancer patients, in which case the amount of tumor DNA is measured relative to wild-type DNA; patients with organ damage or failure, like heart attack, to measure DNA from a particular organ based on specific methylation, relative to total amount of cfDNA. Another relevant application is to investigate forensic samples, such as dried blood spots, saliva, semen and other body fluids and tissues samples from crime scenes, for the presence of minority DNA fractions, which could originate from the perpetrator against background DNA from the victim.

[0056] In another embodiment, there are more than two groups of target DNAs, each group is targeted by several sequence specific assays that generate the same signal. For example, a sample can be analyzed for the presence of beef, pork and horse meat, using multiple assays for beef all having the same one label, multiple assays for pork all having the same second label, and multiple assays for horse all having the same third label. The readout would then distinguish between the three labels very sensitively detecting the three different species.In another embodiment, multiple assays having the same one label are used to detect human chromosome 1, another set of multiple assays have the same second label are used to detect human chromosome two and so forth for each of the 24 human chromosomes (22 somatic, X and Y). This experiment would be a 24-plex.

[0057] Fig. 10 illustrates an embodiment using both labelled and non-labelled control probes for at least one of the N target portions. In another embodiment, only non-labelled control probes are used. The non-labelled control probe competes with the N labelled variant probes for binding to the wild-type sequence of a target portion of the N target portion. Furthermore, the non-labelled control probe will generally outcompete the N labelled variant probes for binding to the wild-type sequence since the non-labelled control probe is complementary to or comprises the wild-type sequence, whereas the N labelled variant probes are complementary to or comprise a sequence variant of a respective target portion of the N target portions and thereby have preference for such a sequence variant over the wild-type sequence. The nonlabelled control probe thereby suppresses non-specific binding of the N labelled variant probes to the wild-type sequence of a target portion of the N target portions.

[0058] In an embodiment, the control probe comprises a 3’ end block. Such a 3’ end block is thereby present at the 3’ end of the control probe to prevent or at least restrict or inhibit extension of the control probe during the PCR amplification in step S2. Any such 3’ end block or modification that can block such extension during PCR could be used including, but not limited to, a 3’ C3 spacer, a 3’ ddNTP, such as 3’ ddC, 3’ inverted dT, 3’ amino group, 3’ phosphorylation and 3’ methylated uracil.

[0059] Fig. 12 illustrates an embodiment of using a non-labelled control probe comprising a 3’ blocking group represented by a 3’ C3 spacer group. The embodiment shown in Fig. 12 corresponds to the one shown in Fig. 7 but with the difference of using a non-labelled control probe instead of a labelled control probe.

[0060] In an embodiment, step S1 comprises contacting the sample with the N primer pairs, the N labelled variant probes, N control probes. In this embodiment, each control probe of the N control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions.

[0061] In this embodiment, each target portion of the N target portions is targeted with a respective labelled variant probe and a respective non-labelled control probe. Such an approach is generally preferred relative merely using a non-labelled control probe for one or a subset of the N target portions since the Nnon-labelled control probes will thereby suppress non-specific binding of the labelled variant probes to the N wild-type sequences of the N target portions.

[0062] In the embodiments comprising the use of one up to N non-labelled control probes, only the target DNA comprising sequence variants will be quantified in step S4 based on the detected first label. In such an embodiment, it may be beneficial to target and quantify one or a few general sites in the target DNA to allow for determining of the amount of DNA in the sample. Such a general site could then be a so-called quantification target or marker that is different from any of the N target portions. In such an embodiment, step S1 preferably comprises contacting the sample with the N primer pairs, the N labelled variant, the non-labelled control probe(s) (one to N non-labelled control probes), a quantification primer pair comprising a forward primer and a reverse primer and a labelled quantification probe. The quantification primer pair is designed to amplify a quantification target of the target DNA and the labelled quantification probe comprises a quantification label different from the first label of the N labelled variant probes. In such an embodiment, step S2 comprises amplifying the N target portions and the quantification target by performing PCR amplification on the target DNA. Step S3 comprises detecting the first label bound to the N amplified target portions or released from the N labelled variant probes and detecting the quantification label bound to the amplified quantification target or released from the labelled quantification probe. Step S4 comprises, in this embodiment, quantifying the target DNA based on the detected first and quantification labels.

[0063] Fig. 13 illustrates a particular embodiment involving, in step S1, contacting the sample with the N primer pairs, the N labelled variant probes, N labelled control probes and N non-labelled control probes. Each labelled control probe of the N labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions and each non-labelled control probe of the N non-labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions. The N labelled control probes have the same second label, and the N control probes preferably comprise a 3’ blocking group.

[0064] In this embodiment, each target portion of the N target portions is targeted with a labelled variant probe, a labelled control probe and a non-labelled control probe. This basically corresponds to Fig. 9 with M=N and complementing each target portion with a non-labelled control probe.

[0065] In a variant, step S1 comprises contacting the sample with the N primer pairs, the N labelled variant probes, N non-labelled control probes and M labelled control probes. In this embodiment, each non-labelled control probe of the N non-labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions and each labelled control probe of the M labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions. The M labelled control proves have the same second label and 1 <M<N, preferably M=N. The N non-labelled control probes preferably comprise a 3’ blocking group.

[0066] Thus, in an embodiment, only one or a subset of the N target portions is targeted with a labelled control probe whereas all N target portions are targeted with labelled variant probes and non-labelled control probes. As an example, for each target portion of the subset of the N target portions, the percentage of the control probes complementary to or comprising the wild-type sequence of the target portion that is labelled is less than 50 %, preferably less than 25 %, more preferably less than 10 %, and most preferably equal to or less than 5 %.

[0067] The method as shown in Fig. 3 and in particular step S2 thereof preferably involves a digital PCR (dPCR) reaction or a quantitative PCR (qPCR), also referred to as real-time PCR.

[0068] In an embodiment, qPCR is used for readout. In an embodiment, one sample is analyzed per reaction container. Duplex reactions are usually performed generating two amplification curves, i.e., one for the first reporter (labelled variant (hydrolysis) probes) and one for the second reporter (labelled control (hydrolysis) probe or labelled quantification (hydrolysis) probe). In such a case, one amplification curve is the combined fluorescence signal from all PCR amplifications of variant sequences having a common first uniprobe, e.g., hydrolysis probes with the same first reporter, and a second amplification curve, which is the combined fluorescence from all PCR amplifications of wild-type sequences having a common second uniprobe, e.g., hydrolysis probes with the same second reporter or the combined fluorescence from all PCR amplifications of the quantification target. But it is possible to run triplex, tetraplex and even higher multiplex to measure the total abundance of multiple groups of target sequences.

[0069] In a second embodiment, dPCR is used for readout. A singleplex reaction can be run targeting, for example, variant sequences known to cause a disease using the same uniprobe for all variants. If uniprobe signal appears, a variant sequence among the targeted was present, although it is not known which one. The total number of positive reactions reflects the minimum number of target DNA molecules harboring a variant sequence that were present and using statistical analysis based on the Poisson distribution the expected number can be estimated. Knowing the total sample volume analyzed, the concentration of target DNA molecules harboring variant sequences can be calculated. Duplex uniprobedPCR can be run targeting variant sequences with one uniprobe and corresponding wild-type sequences or the quantification target with the other uniprobe. The ratio between the number of positive reactions with the two uniprobes reflects the fraction of sequences that are mutated. If the mutant fraction is expected to be low, only a fraction of the uniprobes for wild-type targets are preferably labelled in order to balance the counts of negative and positive reactions. Triplex, tetraplex and even higher multiplex uniprobe dPCR can be run when there are many target groups to quantify, each group represented by at least one but preferably multiple possible targets,

[0070] The invention is, however, not limited to using qPCR and dPCR and may also involve using other PCR amplification methods, such as hyperplex PCR (hpPCR), and other types of amplifications, including, but not limited to, rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), multiple displacement amplification (MDA), helicase dependent amplification (HAD), transcription mediated amplification (TMA). Labelled sequence specific probes bind to the amplified material and may either give rise to signal upon binding, upon dissociation, or upon cleaved (e.g., hydrolysis probes) or when modified during the course of the amplification.

[0071] Complementary as used herein refers both to complete complementarity of nucleotide sequences, in some cases referred to as an identical sequence, as well as complementarity sufficient to achieve the desired binding of nucleotide sequences. Complementary refers to the standard base pairing rules between G-C, A-T and A-U. Certain nucleotides not commonly found in natural nucleotide sequences or chemically synthesized as mentioned in the foregoing may be included in the nucleotide sequences described herein. Complementarity need not be perfect. In clear contrast, stable duplexes may contain mismatched base pairs, degenerative, or unmatched nucleotides. Complementary is characterized by the capacity for precise pairing of purine and pyrimidine bases of one sequence (strand, oligonucleotide) of a nucleic acid molecule to another sequence of a nucleic acid molecule, such that the order of purine and pyrimidine bases matches and binds to (hybridizes with) the other, complementary sequence.

[0072] In the probes, modified bases, nucleoside analogues non-natural bases, altered sugar moieties, analogues sugar moieties, and / or modified or artificial backbones could be used for one or more, including all bases of the probes. Modifications can be different for the different probes. For example, different modifications can be used for variant probes and control probes to thereby moderate their relative affinities. A particular example of a common modification is locked nucleic acid (LNA). It is alsopossible to use different modifications on different variant probes when more than one such variant probe is present.

[0073] The complementarity between the N labeled variant probes and the respective sequence variant of the N target portion and the labelled or non-labelled control probe and the wild-type sequence of a target portion allows the labeled variant probes and labeled or non-labelled control probe to hybridize to the amplified target portions.

[0074] Hybridization or hybridization condition denotes the process in which single-stranded nucleotide sequences anneal to complementary nucleotide sequences. Such annealing between complementary nucleotide sequences is dependent on several parameters including, for instance, ionic strength, temperature, length of the labeled variant or control probes, and G-C-nucleotides content of the variant or control probes.

[0075] The amplification of the N target portions by PCR in step S2 can use any polymerase that is commonly used in PCRs. Illustrative, but non-limiting, examples of such polymerases include Taq polymerase, Pfu polymerase, DNA polymerase I (Klenow fragment), high-fidelity polymerases, such as Phusion DNA polymerase, KAPA HiFi DNA polymerase and Q5 High-Fidelity DNA polymerase, and hot-start polymerases, such as Platinum Taq DNA Polymerase and AccuPrime Taq DNA Polymerase.

[0076] In an embodiment, step S4 comprises quantifying the target DNA based on a ratio between the detected first label bound to the N amplified target portions and the detected second label bound to the N amplified target portions. Thus, in this embodiment, a ratio or quotient between the detected first label and the detected second label is calculated and used to quantify the target DNA in the sample.

[0077] For instance, in a duplex reaction with N labelled variant probes targeting variant sequences and a labelled control probe targeting wild-type sequences, the ratio between the concentrations reflected by the two labels reflects the ratio of variant to wild-type sequences. For example, if qPCR is used and a Cq(WT) and Cq(variant) are measured then the ratio is: 2<Cci(WT)-Cci(variant)),Sjncethe crossing points of the amplification curves with the threshold lines are proportional to the logarithm of the initial number of targeted molecules. When dPCR is used the expected number of wild-type target and variant targets are calculated from the numbers of positive wild-type and variant dPCR reactions corrected for Poisson distributions as known in the field. In both cases, it is possible to have only a fraction of control probe labelled. This reduces the signal from those wild-type targets, which can be advantageous if the group isdominant. When calculating the ratio of the two targeted groups, such fractional labelling must be considered.

[0078] In an embodiment, step S1 in Fig. 3 comprises contacting the sample with the N primer pairs PPi each comprising a forward primer FP, and a reverse primer RPi, the N labelled variant probes VP, and the control probe CP. In this embodiment, / =1...N and N is an integer equal to or larger than 2. Each primer pair PPi of the N primer pairs is designed to amplify a respective target portion TPi of the target DNA. Each labelled variant probe VP, of the N labelled variant probes is complementary to a sequence variant of a respective target portion TPi of the N target portions and the control probe CP is complementary to a wild-type sequence of a target portion TP] of the N target portions, and j e [1 , A / |.

[0079] The number N is, as mentioned above, an integer equal to or larger than 2. In a preferred embodiment, N is an integer equal to or larger than 3, preferably equal to or larger than 5, more preferably equal to or larger than 10, and most preferably equal to or larger than 15.

[0080] In the above-described embodiment, a single control probe is used to target the wild-type sequence of one of the target portions of the DNA target. The invention is, however, not limited thereto. Generally, the sample can be contacted with k control probes in step S1 and there this number k e[1, / V], i.e., is an integer from one up to N.

[0081] For highest specificity, competition between the control probes and the variant probes is desired for all target portions of the target DNA and not only one of the target portions (target 1 in Fig. 7). Hence, in an embodiment, step S1 comprises contacting the sample with the N primer pairs, the N labelled variant probes, N labelled control probes, wherein each labelled control probe of the N labelled control probes is complementary to a wild-type sequence of a respective target portion of the N target portions. Further, each labelled control probe of the N labelled control probes comprises the second label.

[0082] In such an embodiment, each target portion is interrogated with a respective labelled variant probe and a respective labelled control probe. However, depending on the particular target DNA in the sample, such an approach might produce too large wild-type signal, i.e., too high signal for the second label as compared to the first label. In order to reduce the signal from the labelled control probes but still maintain the highest specificity only a fraction of the control probes is labelled.In such an embodiment, step S1 comprises contacting the sample with the N primer pairs, the N labelled variant probes, N non-labelled control probes and the labelled control probe. In this embodiment, each non-labelled control probe of the N non-labelled control probes is complementary to or comprises a wildtype sequence of a respective target portion of the N target portions. The N non-labelled control probes thereby lack any label and are thereby unlabeled control probes.

[0083] In an embodiment, the N labelled variant probes, the N non-labelled control probes and the labelled control probe lack 3’ OH group or have a 3’ end block inhibiting probe extension. Such a 3’ end block or the lack of 3’ OH group thereby means that the probes do not extend during the PCR amplification in step S2.

[0084] There are various 3’ end blocks or modification that can be used to prevent probe extension during PCR including, but not limited to, 3’ dideoxycytidine (ddC), 3’ inverted dT, 3’ 03 spacer, 3’ amino, 3’ phosphorylation and 3’ methylated uracil.

[0085] In the above-described embodiment, unlabeled control probes are used for each of the N target portions of the target DNA but merely a single labeled control probe is used.

[0086] In another embodiment, step S1 comprises contacting the sample with the N primer pairs, the N labelled variant probes, N non-labelled control probes and M labelled control probes. In this embodiment, each non-labelled control probe of the N non-labelled control probes is complementary to or comprises a wildtype sequence of a respective target portion of the N target portions. Each labelled control probe of the M labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions. The M labelled control probes have a same second label and 1<M<N.

[0087] In this embodiment, N unlabeled control probes are used together with M labelled control probes in addition to the N primer pairs and the N labelled variant probes. The number of labelled control probes, i.e. , M, is equal to or lower than the number of target portions in the target DNA, i.e. , N. In a preferred embodiment, M=N. The N unlabeled control probes are preferably 3’ end blocked to inhibit primer extension during the PCR amplification in step S2. The various examples of 3’ end blocks mentioned above can also be used for the N control probes in this embodiment.

[0088] In an embodiment, the ratio of labeled control probe vs. unlabeled control probe is, per target portion, preferably lower than 1, i.e., the amount of unlabeled control probe complementary to a particular wild-type sequence of a target portion of the target DNA is preferably higher than the amount of labeled control probe complementary to the particular wild-type sequence.

[0089] In various embodiments, N is equal to or larger than 3, preferably equal to or larger than 4, such as equal to or larger than 5, equal to or larger than 6, equal to or larger than 7, equal to or larger than 8, such as equal to or larger than 9 or equal to or larger than 10. In these embodiments, M is preferably 1 or 2. Hence, it is generally sufficient to have 1 or 2 labeled control probes when used together with, for instance, 3-10, or indeed even more, unlabeled control probe.

[0090] In an embodiment, for each target portion of the N target portions, the percentage of the control probes complementary to or comprising the wild-type sequence of the target portion that is labelled is less than 10 %. In a preferred embodiment, the percentage of the control probes complementary to or comprising the wild-type sequence of the target portion that is labelled is less than 5 %, preferably less than 2.5 %, and more preferably equal to or less than 1 %. In a preferred embodiment, the percentage of the control probes complementary to or comprising the wild-type sequence of the target portion that is labelled is selected within an interval of from 0.01 up to 1 %.

[0091] Such an approach maintains the competition between the control probes and the variant probes for achieving high specificity but avoids the risk of having too high signal of the second label as compared to the signal of the first label in step S3.

[0092] In a further embodiment, step S1 comprises contacting the sample with the N primer pairs, the N labelled variant probes, Mi non-labelled control probes and M2 labelled control probes. In this embodiment, each no n-l abel led control probe of the Mi non-labelled control probes is complementary to or comprises a wildtype sequence of a respective target portion of the N target portions. Each labelled control probe of the M2 labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions. The M2 labelled control probes have a same second label and 1<Mi<N and 1<M2^N.

[0093] In a particular embodiment, M2<MI.

[0094] In another particular embodiment, M +M2=N. In this particular embodiment, each non-labelled control probe of the Mi non-labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of a first subset consisting of Mi target portions of the N target portions and eachlabelled control probe of the M2 labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of a second subset consisting of M2 target portions the N target portions. The first subset consists of Mi target portions of the N target portions whereas the second subset consists of the remaining M2=N-M target portions of the N target portions. This means that all sequence variants of the N target portions are targeted by a respective labelled variant probe whereas Mi of the corresponding N wild-type sequences are targeted by non-labelled control probes and the remaining M2 wild-type sequences are targeted by labelled control probes.

[0095] In an example embodiment, M2=1 and A i=A / -1.

[0096] Various types of labels that can be used as the first label of the N labelled variant probes and the second label of the labelled control probe or the M labelled control probes, and the quantification label of the labelled quantification probe. Illustrative, but non-limiting, examples of such labels include fluorophores, radioactive labels, enzymatic labels, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), and chemiluminescent labels.

[0097] In a preferred embodiment, the first and second labels are a first and second fluorophore.

[0098] Illustrative, but non-limiting, examples of fluorophores include fluorescein, carboxyfluorescein (FAM), such as 6-FAM or 5-FAM, 4,5-dichloro-2',7'-dimethoxyfluorescein (JOE), tetrachlorofluorescein (TET), Cal Fluor® Gold 540, Cal Fluor® Orange 560, tetramethylrhodamine (TAMRA), Cyanine 3, Quasar® 570, Cal Fluor® Red 590, carboxy-X-Rhodamine (ROX), sulforhodamine 101-X, Cyanine 5, Quasar 670, Cyanine 5.5, VIC, and hexachlorofluorescein (HEX). As an example, the first label could be 6-FAM with the second label being HEX.

[0099] Fig. 4 illustrates an embodiment of steps S1 and S2 in Fig. 3. In this embodiment, step S1 comprises contacting, in step S10, the sample with the N primer pairs each comprising a forward primer, also referred to as preamplification forward primer herein, having a first adapter sequence and a reverse primer, also referred to as preamplification reverse primer herein, having a second adapter sequence. Step S2 comprises, in this embodiment, amplifying, in step S11, the N target portions by performing PCR pre-amplification with less than ten cycles to form the N amplified target portions. Step S1 also comprises, in this embodiment, contacting, in step S12, the sample following the PCR-pre-amplification with an amplification forward primer having a sequence corresponding or complementary to the first adapter sequence and an amplification reverse primer having a sequence corresponding or complementary tothe second adapter sequence, the N labelled variant probes and the control probe. Step S2 also comprises, in this embodiment, amplifying, in step S13 and following contacting the body fluid sample with the amplification forward primer, the amplification reverse primer, the N labelled variant probes and the control probe, the N amplified target portions by performing PCR amplification on the N amplified target portions.

[0100] This embodiment thereby performs a PCR pre-amplification in step S11 for a limited number of PCR cycles, i.e., less than ten cycles. In an embodiment, the PCR pre-amplification is performed for less than five cycles, preferably less than three cycles.

[0101] In an embodiment, the first adapter sequence and the second adapter sequence are the same or complementary to each other. Such an approach simplifies the design of the amplification forward and reverse primers.

[0102] In an embodiment, melting temperatures of the preamplification forward primers and of the preamplification reverse primers are lower than melting temperatures of the amplification forward primers and of the amplification reverse primers. In such an embodiment, the PCR pre-amplification in step S11 is done at a temperature that is preferably below the melting temperatures of the preamplification forward primers and of the preamplification reverse primers. The PCR amplification in step S13 is done at a comparatively higher temperature, preferably above the melting temperatures of the preamplification forward primers and of the preamplification reverse primers but below the melting temperatures of the amplification forward primers and of the amplification reverse primers. In an embodiment, the PCR preamplification in step S11 and the PCR amplification in step S13 are performed in the same reaction vessel.

[0103] Thus, in a preferred embodiment, the PCR pre-amplification in step S11 is performed at a lower temperature than the PCR amplification in step S13. This means that the N primer pairs comprising a respective preamplification forward primer and a respective preamplification reverse primer will, at the lower temperature of the PCR pre-amplification in step S11, be able to bind to the N target portions of the target DNA and thereby amplify these N target portions in step S11. The temperature is then raised above the melting temperature of the preamplification forward and reverse primers to thereby allow the amplification forward and reverse primers to bind to the N amplified target portions and amplify the same in step S13.In an embodiment, a PCR pre-amplification step is included with target specific primers flanked with generic sequences that serve as primers in the subsequent PCR. Either two generic sequences, one to each primer, or a single generic sequence to both primers, can be introduced. Pre-amplification is run a small number of cycles only, preferably five or less, more preferably three or less and most preferably a single round or two rounds only. In the subsequent PCR, primers binding the introduced flanking sequences can be used. A single primer is sufficient if the same generic flanking sequences were added to both the upstream and downstream primer, or two generic primers are used. The advantage of introducing the pre-amplification step is to reduce sequencing bias in the subsequent PCR. In an embodiment, the PCR pre-amplification in step S11 and the PCR amplification in step S13 are performed in the same reaction vessel.

[0104] Fig. 11 illustrates an embodiment involving the use of target specific primers with common overhangs with sequence A for all upstream primers and sequence B for all downstream primers. The overhangs extend the targeted portion with the A and B sequences and their complements in the opposite strand. In an embodiment, these primers are used in the pre-amplification, which preferably involves only two or just a few initial PCR cycles, whereafter they are replaced by generic primers having sequences A and B. Bias is thereby reduced since all target sequences are amplified with the same primer pair in these subsequent cycles. Overhang sequences A and B may be the same or can be different. If they are the same, a single primer can be used to amplify all target sequences in the subsequent cycles following the incorporation of the overhangs.

[0105] Ultraviolet (UV) radiation (UVR) or light induces mutations at certain positions in the genome that are highly vulnerable to mutagenesis by UV light (Elliott et al., Elevated pyrimidine dimer formation at distinct genomic bases underlies promoter mutation hotspots in UV-exposed cancers, PLoS Genetics (2018) 14(12): e1007849; Mao et al., ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma, Nature Communications (2018) 9(1): 2626; Elliot et al., Mechanistic basis of atypical TERT promoter mutations, Nature Communications (2024) 15(1): 9965; Selvam et al., Detecting recurrent passenger mutations in melanoma by targeted UV damage sequencing, Nature Communications (2023) 14(1): 2702). Specifically, these mutations have a characteristic sequence signature, primarily cytosine (C) to thymine (T) substitutions in dipyrimidine sequence contexts, which arise due to the main mutagenic mechanism of UV light involving formation of DNA damage in the form of cyclobutene pyrimidine dimers (CPDs). Some proteins, when bound to genomic DNA, can alter the local propensity to DNA damage from UV light. In particular, transcription factors of the E26 transformation specific (ETS) family, when bound to their specific binding sites or elements, can potentlyincrease CPD formation in response to UV light. Increased damage formation at ETS transcription factor binding sites occurs primarily at pyrimidines immediately upstream of the core motif (YYTTCCK) but also, to a lesser extent, at the central dipyrimidine position within the core motif (TTCCK). This phenomenon leads to widespread recurrent mutation hotspots in skin cells at such ETS transcription factor binding sites and sharply elevated mutation rate at such hotspot sites. In agreement with these ETS transcription factor binding sites being hypersensitive specifically to UV light, ETS hotspots mutations are common in sun-exposed skin cancers as well as normal skin cells, while being completely absent in other non-UV exposed tissue (Fredriksson et al., Recurrent promoter mutations in melanoma are defined by an extended context-specific mutational signature, PLoS Genetics (2017) 13(5): e1006773). Importantly, mutations at ETS transcription factor binding sites are exclusively taking place in cells that have been directly exposed to UV light and it is therefore highly unlikely that these ETS transcription factor binding sites are mutated in cells that have not been exposed to UV light. This means that mutations in such ETS transcription factor binding sites in cfDNA can be used as an indication that the cfDNA is of skin origin, such as being skin-derived ctDNA, or has been exposed to UV light for other reasons.

[0106] In an embodiment, the N target portions are ETS transcription factor binding sites. In such an embodiment, the N sequence variants are mutations at the N ETS transcription factor binding sites and the wild-type sequence is a non-mutated wild-type sequence of an ETS transcription factor binding site of the N ETS transcription factor binding sites.

[0107] The method of the invention can then be used to quantify the fraction of circulating free DNA (cfDNA) that is skin derived from a liquid biopsy, such as a blood sample, by quantifying the amount of mutations at the N ETS transcription factor binding sites. Thus, cfDNA that comprises mutations at ETS transcription factor binding sites are derived from cells that have been exposed to UV light, i.e. , skin cells. This means that cfDNA originating from skin cells or cells of skin origin then comprise such mutations at ETS transcription factor binding sites. Accordingly, the present invention can be used to quantify the fraction of cfDNA that is skin derived. This is possible that the likelihood of having any mutation at the ETS transcription factor binding sites is extremely low for cells not exposed to UV light, i.e., non-skin cells. This means that the cells of non-skin origin typically have the wild-type rather the mutated sequences of the ETS transcription factor binding sites.

[0108] In an embodiment, the target DNA is a circulating tumor DNA (ctDNA) and the sample is a body fluid sample or the target DNA is tumor DNA and the sample is a tumor sample. In such an embodiment, themethod further comprising an additional step S5 as shown in Fig. 5. This step S5 comprises estimating a tumor mutational burden based on the quantified ctDNA or the tumor DNA.

[0109] In this embodiment, the amount of ctDNA or tumor DNA is quantified using a quantifying of mutations at ETS transcription factor binding sites. This also means that a tumor mutational burden can be estimated based on the quantified ctDNA or tumor DNA, i.e., based on the quantified mutations at the ETS transcription factor binding sites. A high quantity of mutations at the ETS transcription factor binding sites as compared to the wild-type sequences at these ETS transcription factor binding sites implies a high tumor mutational burden. This provides valuable diagnostics information of the tumor from the patient and can then be used as a basis when selecting, for instance, treatment strategy, recurrence, and / or patient surveillance schedule.

[0110] Instead of using mutations at ETS transcription factor binding sites to quantify ctDNA and tumor mutational burden, the patient’s tumor DNA can be sequenced in order to detect so called private mutations, i.e., mutations present in the tumor DNA but not present in the genomic DNA from non-cancerous cells from the patient. In such a case, the target portions could include sequences in the genomic DNA comprising such detected private mutations with the N sequence variants representing the private mutations and the wild-type sequences corresponding to the non-mutated genomic sequences in these target portions.

[0111] In an embodiment, the target DNA is a target cfDNA and the sample is a body fluid sample. Thus, a preferred embodiment, of a sample is a body fluid sample that can contain cfDNA. Illustrative, but nonlimiting, examples of such body fluid samples include blood sample, serum sample, plasma sample, urine sample, cerebrospinal fluid (CSF) sample, saliva sample, amniotic fluid sample, pleural effusion sample, breast milk sample, and a sputum sample.

[0112] In an embodiment, the target cfDNA is skin-derived cfDNA. In such an embodiment, the N target portions of the skin-derived cfDNA are ETS transcription factor binding sites. The N sequence variants are mutations at the N ETS transcription factor binding sites and the wild-type sequence is a non-mutated wild-type sequence of an ETS transcription factor binding site of the N ETS transcription factor binding sites.

[0113] In another embodiment, the target cfDNA is circulating donor DNA. The N target portions of the circulating donor DNA represent N single nucleotide polymorphisms (SNPs) for which a donor and a recipient areboth homozygotes but have reciprocal alleles. The N sequence variants are N donor-specific SNP alleles and the wild-type sequence is a recipient-specific SNP allele.

[0114] The invention can, thus, be used to monitor a recipient of transplanted organ, such as lung, kidney, liver, and heart. Genotyping or sequencing of both the donor and recipient is used to identify SNPs where the donor and recipient are both homozygotes but have reciprocal alleles. Assays targeting those SNPs distinguish donor DNA from recipient DNA. This means that the invention can be used to quantify the donor cfDNA in a body fluid sample from a recipient. Such a quantification can be useful to monitor the engraftment process and the health of the transplanted organ.

[0115] In a further embodiment, the target cfDNA is ctDNA. In this embodiment, the N target portions of the ctDNA are N cancer hotspot genomic portions. The N sequence variants are cancer-inducing mutations at the N cancer hotspot genomic portions and the wild-type sequence is a non-mutated wild-type sequence of a cancer hotspot genomic portion of the N cancer hotspot genomic portions.

[0116] Cancer hotspots in genomic DNA are specific regions that are more prone to mutations, which can drive the development and progression of cancer. These hotspots often occur in genes that are critical for cell growth, division, and survival. Hotspots are frequently found in oncogenes (genes that promote cell growth) and tumor suppressor genes (genes that inhibit cell growth). Examples include mutations in the TP53, BRCA1 , BRCA2, KRAS, and BRAF genes. Cancer hotspots could be recurrent mutations meaning that the same mutations are found in many different cancer patients. This recurrence suggests that these mutations provide a growth advantage to cancer cells.

[0117] In an embodiment, the target DNA is circulating tumor DNA (ctDNA) and the N target portions of the ctDNA are N portions that are mutated in the tumor or have a variant sequence that is not a germline variant.

[0118] In an embodiment, the sample is a liquid biopsy from a recipient having a transplanted organ from a donor. In this embodiment, the target DNA is circulating cell free DNA (cfDNA) and the N target portions of the cfDNA are N portions harboring a sequence variation, where both the donor and recipient are homozygotes but with different alleles.

[0119] In an embodiment, the target DNA is target complementary DNA (cDNA) obtained by reverse transcription of ribonucleic acid (RNA) in a body sample.In an embodiment, a first set of variant probes with a first label is used to bind to target portions in the target DNA in a first sample, such as a sample from a first subject, and a second set of variant probes with a second, different label is used to bind to target portions in the target DNA in a second sample, such as a sample of a second subject. In such a case, the samples can be pooled and still be detected in a duplex measurement, thereby characterizing the two samples in a single run, leading to cost savings. This process can be extended further to pool more than two samples and using more than two sets of variant probes with different labels.

[0120] In the above-described embodiments, the N variant probes and control probe(s) are oligonucleotide probes comprising the first label or the second label in the case of labelled control probe(s). In another embodiment, the N variant probes and the control probe(s) are padlock probes. Such an embodiment is shown in Fig. 6. The method comprises contacting, in step S20, the sample with N primer pairs each comprising a forward primer and a reverse primer, N variant padlock probes and a control padlock probe. N is an integer equal to or larger than 2. Each primer pair of the N primer pairs is designed to amplify a respective target portion of the target DNA. Each variant padlock probe of the N variant padlock probes is complementary to or comprises, preferably complementary to, a sequence variant of a respective target portion of the N target portions and the control padlock probe is complementary to or comprises, preferably complementary to, a wild-type sequence of a target portion of the N target portions. The method also comprises joining, in step S21, 5’ and 3’ ends of padlock probes of the N variant padlock probes and the control padlock probe while hybridized to the target DNA to form circular variant padlock probes and circular control padlock probes. The method further comprises rolling circle amplifying (RCA), in step S22, the circular variant padlock probes with first labelled amplification primers having a first label and complementary to a portion of the circular variant padlock probes to generate first labelled rolling circle products (RCPs) and the circular control padlock probes with second labelled amplification primers having a second label different from the first label and complementary to a portion of the circular control padlock probes to generate second labelled RCPs. The first labelled RCPs and the second RCPs are detected in step S23 and the following step S24 comprises quantifying the target DNA based on the detected first and second labelled RCPs.

[0121] Padlock probe as used herein refers to any probe capable of being circularized following hybridization to a target DNA.Joining the 5’ and 3’ ends of padlock probes of the N variant padlock probes and the control padlock probe while hybridized to the target DNA could be a direct join of the 5’ and 3’ ends or an indirect join of the 5’ and 3’ ends.

[0122] In an embodiment, step S21 comprises ligating the 5’ and 3’ ends of padlock probes of the N variant padlock probes and the control padlock probe while hybridized to the target DNA. In such an embodiment, a ligating agent is used to ligate the 5’ and 3’ ends of the padlock probes. In an embodiment, the ligating agent is a ligase, and in particular a DNA ligase. A ligase is an enzyme that facilitates the joining of nucleotide strands together by catalyzing the formation of a phosphodiester bond. Any ligase capable of ligating together the 5’ and 3’ ends of the padlock probes can be used according to the embodiments.

[0123] In a particular embodiment, the ligating agent is a thermostable ligating agent, preferably a thermostable ligase, and in particular a thermostable DNA ligase. For instance, the thermostable ligase could be selected from the illustrative group comprising Ampligase® DNA ligase, Taq DNA ligase, Pfu DNA ligase and 9°N™ DNA ligase. Other non-limiting, but illustrative, examples of DNA ligases include Chlorella virus DNA ligase, also referred to as Paramecium bursaria Chlorella virus 1 (PBCV-1) DNA ligase or SplintR ligase, Escherichia coli DNA ligase encoded by the lig gene; T4 or T7 DNA ligase from bacteriophage T4 or T7; DNA ligase I, II, III or IV.

[0124] Optionally, step S21 comprises adding a ligating agent, preferably a ligase, more preferably a DNA ligase, to the sample to ligate together the 5’ and 3’ ends of the padlock probes.

[0125] In an embodiment, the 5’ end of the padlock probes comprises a 5’ phosphate group to facilitate ligation of the 5’ and 3’ ends using the ligating agent.

[0126] In other embodiments, the padlock probes hybridize to the target DNA with a gap between the 5’ and 3’ ends. The joining of the 5’ and 3’ ends in step S21 is then an indirect joining of these ends. In such a case, the gap between the 5’ and 3’ ends is filled with at least one gap-filling oligonucleotide or molecular inversion probe that is capable of binding to the target DNA. Alternatively, or in addition, the gap can be filled by extension of the 3’ end of the padlock probes while hybridized to the target DNA. In such a case, the target DNA acts as a template for the extension with the padlock probe acting as primer. Such an extension is performed by a polymerase and nucleotides (dNTPs) added to the sample. In the abovedescribed embodiments, a ligating agent, such as the above-described ligases, could join the 5’ and 3’ ends and the gap-filling oligonucleotide(s) or the 5’ end and the extended 3’ end.Optionally, the polymerase used for 3’ end extension could be inactivated prior to rolling circle amplification in step S22. Such a polymerase inactivation can be performed according to well-known techniques including, but not limited to, heat inactivation.

[0127] The formed circular variant padlock probes and circular control padlock probes are then amplified in step S22 by so-called RCA using first labelled amplification primers to generate the first labelled RCPs and using the second labelled amplification primers to generate the second labelled RCPs. RCA uses a strand-displacing polymerase to extend the labelled amplification primers hybridized to the circular padlock probes. The strand displacing activity of the polymerase displaces the extended labelled amplification primers effectively causing the circular padlock probes to “roll” during RCA.

[0128] Illustrative, but non-limiting, examples of strand-displacing polymerases that could be used in step S22 include Phi29 DNA polymerase, Bst polymerase, Klenow fragment, and derivatives thereof.

[0129] In an embodiment, padlock probes are used for readout, which preferably are rolling circle amplified (RCA) detecting the RCA product (RCP) by one of several means, including binding fluorescent or colored probes, beads etc. In a singleplex setup, uniprobes or unibeads, all with the same color, are used to detect a group of target sequences, such as all variant sequences that can cause a disease. A duplex setup with one uniprobe targeting all variant sequences and a second uniprobe to target corresponding wild-type sequences. Also here only a fraction of the uniprobes for wild-type sequences can be labelled to balance the signals from the uniprobe reactions. Triplex, tetraplex and higher multiplex uniprobe reactions can be run to detect groups of targets each represented by multiple sequences. Examples of high multiplex reactions can be detection of viral, bacterial and fungal species, or mutations in a pathogen that confer resistance to a particular treatment. Similarly, genetic sequence variants can be grouped that should receive a particular treatment for diseases like cancer. Uniprobe as used herein relates to a group of probes that differ in their target sequence by have a common or same reporter, i.e., label.

[0130] The various embodiments discussed in the foregoing also apply to the embodiment shown in Fig. 6.

[0131] EXAMPLE

[0132] Materials and MethodsdPCR primer design and assay conditions

[0133] Primers (Table 1) and probes (Table 2) were designed with Primer3 to target specific UV hotspots detected in the melanoma patients to specifically distinguish between the mutant and wild-type sequences. To test the specificity of the probes, gBIocks (130-135 bp) were designed centered on the probe sequence for each mutant and wild-type probes (Table 3). 54,000 copies of the gBIock were used to maximize the number of positive partitions in the QIAcuity Probe dPCR assay using a QIAcuity Nanoplate 26k 24-well plate. A primer-probe mix containing 0.8 pM forward primer, 0.8 pM reverse primer and 0.4 pM probe was used for all assays, including the multiplex assays.

[0134] Table 1. Primer sequences

[0135]

[0136] Amplicon shows hg38 coordinates.

[0137] Table 2. Probes

[0138]

[0139] Bold and underlined bases indicate the specific base in each probe, where C / G is wild-type (HEX) and T / A is mutant (6-FAM). Hotspot position (hg38) is indicated in the probe name.Table 3. gBIock sequences

[0140]

[0141] g Block sequences containing the specific wild-type (WT) and mutant (MUT) sequences for each hotspot. Specific base is highlighted in bold and underlined. Sequence (ATAT) in italics is an artificial sequence used for ERGIC3 gBIock to decrease the high GC content and allow DNA synthesis. Amplicon size (bp) is indicated.Table 4. Blockers

[0142]

[0143] UV-treated A375 DNA, which results in the generation of low-level mutations at UV-hotpots, was then used to test the sensitivity of the probes with decreasing amounts of UV mutations. A375 cells were treated, or not, five times a week with a dose of 36 J / m2ultraviolet C (UVC), roughly equivalent to 6 hours in the sun. Cells were cultured with daily UV treatment for 6 weeks before harvesting the DNA using a QIAgen Blood and Tissue mini kit. For the dPCR, UV treated DNA was diluted with non-UV treated DNA to dilute out the UV treated DNA. Dilutions used were 100%, 33% ,10% and 0% UV DNA. To determine whether multiplexing of additional assays with the same fl uorophore increased sensitivity of detecting UV damage, 5-plex, 3-plex and single plex assays were used. 20 ng gDNA from the UV / non-UV mixtures were digested with 0.25 U / pl EcoRI prior to assay using the QI Acuity PCR Probe mix and QIAcuity Nanoplate 8.5k 24 well plates. A primer-probe mix containing 0.8 pM forward primer, 0.8 pM reverse primer and 0.4 pM probe was used for all assays.

[0144] For RPL13A single and double probe reactions, 70 ng UV or non-UV DNA was used with the same reaction conditions as above. For 10-plex and 5-plex reactions, 20 ng dilutions at 100%, 50% ,10% and 0% UV DNA and QIAcuity Multiplex Probe mix was used with a C3-spacer containing blocker added at 20-fold excess to the mutant probe (0.8 pM forward primer, 0.8 pM reverse primer, 0.4 pM mutant probe and 8 pM blocker).Results

[0145] dPCR probes specifically identify mutations in artificial DNA

[0146] To determine whether specific probes could distinguish between wild-type and mutant sequences, dPCR experiments were performed with artificial DNA gBIocks first in single-plex assays. Here, positive partitions were limited to the respective gBIocks, as mutant probes displayed positive partitions for mutant gBIocks but not wild-type gBIocks and vice versa (Fig. 1). A handful of positive partitions detected in some conditions, which may indicate sequencing errors during artificial DNA synthesis, seen only for the CSPP1 and RPL29 assays. The 5 assays (CHCHD2, CSPP1, ERGIC, RPL15, RPL29) were then combined into a 5-plex assay, maintaining the same probe and primer concentrations, and again tested for specificity between the mutant and wild-type gBIocks. Here the 5-plex assay showed similar values to the single plex, confirming that these probes can be used in a multiplex.

[0147] Fig. 1 illustrates 1 D scatterplots showing the positive partitions for a given assay (contains both the wildtype HEX and mutant FAM probes for the indicated hotspots) in (Fig. 1A) 1 -plex and (Fig. 1C) 5-plex assays, where all 5 assays were added in a single well. Line indicates positive partition detection threshold. The number of positive partitions is plotted for (Fig. 1B) 1 -plex, and (Fig. 1D) 5-plex assays. Positive partitions were only detected for the wild-type-wild-type or mutant-mutant gBIocks as expected. No detectable difference could be seen for the 1 -plex vs 5-plex assays.

[0148] Multiplex probes specifically detect low levels of UV mutations in UV-treated A375 cell DNA

[0149] In order to test the sensitivity of the assays in detecting low levels of UV damage, UV-treated A375 DNA, which generates mutations at UV-hotspots at a subclonal rate (less than 5% allele frequency) was diluted further with non-UV exposed DNA from the same cells. This DNA was then assayed using the 5-plex assay (CHCHD, CSPP1, ERGIC, RPL15, RPL29) and compared to a 3-plex assay (RPL15, ERGIC3, CSPP1) and a 1 -plex assay (RPL15). Here the 5-plex assay showed an increased number of positive partitions suggesting that combining multiple assays with the same fluorophore led to increased sensitivity in detecting UV-induced mutations in these samples (Fig. 2).

[0150] Double mutant dPCR probes increase sensitivity without increasing assay complexity

[0151] Each ETS binding site can be mutated at multiple positions. Accordingly, mutant probes were designed to target two of these mutations at once. In Fig. 14, we demonstrate that such a double mutant probe, targeting an ETS site in the RPL13A promoter, provides increased sensitivity when applied to UV-exposed DNA compared to probes targeting single mutation, without increasing the number of probes.Competitor oligos maybe used in place of wildtype probes in a multiplex assay

[0152] In another version of the assay, we exchanged the wildtype probes for competitor oligonucleotides, containing 3' blocking C3 spacer groups, replacing the hydroxyl group. This avoids the need for wildtype probes, although in such an assay it may be useful to target and quantify one or a few general genomic sites to allow for determining of the amount of input DNA. Results from a multiplex assay targeting the ETS sites previously shown in Fig. 2a, but using competitor oligonucleotides, applied to UV-exposed DNA is shown as 5-plex-1 assay in Fig. 15A with mutant probes according to SEQ ID NO: 59-63 and competitor oligonucleotides according to SEQ ID NO: 49-53. Five other targets with double mutations, RPL13A, SMUG1, PDCD11, ASXK2, DPH3, were also tested in multiplex using competitor oligonucleotides as 5-plex-2 (Fig. 15b). Finally, a 10-plex assay competitor oligonucleotide assay was tested by combining these two 5-plex assays (Fig. 15c).

[0153] The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible.

Claims

CLAIMS1. A method for quantifying a target deoxyribonucleic acid (DNA) in a sample, the method comprising:contacting (S1) the sample with N primer pairs each comprising a forward primer and a reverse primer, N labelled variant probes and a control probe, whereinN is an integer equal to or larger than 2;each primer pair of the N primer pairs is designed to amplify a respective target portion of the target DNA;each labelled variant probe of the N labelled variant probes is complementary to or comprises a sequence variant of a respective target portion of the N target portions;the control probe is complementary to or comprises a wild-type sequence of a target portion of the N target portions; andthe N labelled variant probes have a same first label;amplifying (S2) the N target portions by performing polymerase chain reaction (PCR) amplification on the target DNA;detecting (S3) the first label bound to the N amplified target portions or released from the N labelled variant probes; andquantifying (S4) the target DNA based on the detected first label.

2. The method according to claim 1, wherein contacting (S1) the sample comprises contacting (S1) the sample with the N primer pairs, the N labelled variant probes, N control probes, wherein each control probe of the N control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions.

3. The method according to claim 1 or 2, wherein the control probe is an non-labelled control probe comprising a 3’ end block.

4. The method according to claim 1 or 2, whereinthe control probe is a labelled control probe having a second label different from the first label; detecting (S3) the first label comprises detecting (S3) first and second labels bound to the N amplified target portions or released from the N labelled variant probes or the labelled control probe; and quantifying (S4) the target DNA comprises quantifying (S4) the target DNA based on the detected first and second labels.

5. The method according to claim 4, wherein quantifying (S4) the target DNA comprises quantifying (S4) the target DNA based on a ratio between the detected first label and the detected second label.

6. The method according to claim 4 or 5, wherein contacting (S1) the sample comprises contacting the sample with the N primer pairs, the N labelled variant probes, N non-labelled control probes, and N labelled control probes, whereineach labelled control probe of the N labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions;each non-labelled control probe of the N non-labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions;the N labelled control probes have the same second label; andoptionally the N control probes comprise a 3’ blocking group.

7. The method according to claim 4 or 5, wherein contacting (S1) the sample comprises contacting the sample with the N primer pairs, the N labelled variant probes, N non-labelled control probes and M labelled control probes, whereineach non-labelled control probe of the N non-labelled_control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions;each labelled control probe of the M labelled control probes is complementary to or comprises a wild-type sequence of a respective target portion of the N target portions;the M labelled control proves have the same second label;1 <M<N, preferably M=N andoptionally the N non-labelled control probes comprise a 3’ blocking group.

8. The method according to claim 7, wherein for each target portion of the N target portions, the percentage of the control probes complementary to or comprising the wild-type sequence of the target portion that is labelled is less than 10 %, preferably less than 5 %, more preferably less than 2.5 %, and most preferably equal to or less than 1 %, such selected within an interval of from 0.01 up to 1 %.

9. The method according to any one of claims 4 to 8, wherein the first label is a first fluorophore and the second label is a second, different fluorophore.

10. The method according to any one of claims 4 to 8, whereineach labelled variant probe of the N labelled variant probes comprises a first reporter and a first quencher; andthe labelled control probe comprises a second reporter different from the first reporter and a second quencher or the first quencher.

11. The method according to any one of claims 1 to 10, whereincontacting (S1) the sample comprises contacting (S10) the sample with the N primer pairs each comprising a forward primer having a first adapter sequence and a reverse primer having a second adapter sequence;amplifying (S2) the N target portions comprises amplifying (S11) the N target portions by performing PCR pre-amplification with less than ten cycles, preferably less than five cycles, more preferably less than three cycles of the target DNA to form the N amplified target portions;contacting (S1) the sample further comprises contacting (S12) the sample following the PCR-pre-amplification with an amplification forward primer having a sequence corresponding or complementary to the first adapter sequence and an amplification reverse primer having a sequence corresponding or complementary to the second adapter sequence, the N labelled variant probes and the control probe; andamplifying (S2) the N target portions further comprises amplifying (S13), following contacting the sample with the amplification forward primer, the amplification reverse primer, the N labelled variant probes and the control probe, the N amplified target portions by performing PCR amplification on the N amplified target portions.

12. The method according to claim 11, wherein the first adapter sequence and the second adapter sequence are the same or complementary to each other.

13. The method according to claim 11 or 12, whereinmelting temperatures of the forward primers and of the reverse primers are lower than melting temperatures of the amplification forward primers and of the amplification reverse primers;the PCR pre-amplification is done at a first temperature, preferably below the melting temperatures of the forward primers and of the reverse primers; andthe PCR amplification is done at a second temperature, higher than the first temperature, preferably above the melting temperatures of the forward primers and of the reverse primers but below the melting temperatures of the amplification forward primers and of the amplification reverse primers;optionally wherein the PCR pre-amplification and the PCR amplification are performed in the same reaction vessel.

14. The method according to any one of claims 1 to 13, whereinthe N target portions are E26 transformation-specific (ETS) transcription factor binding sites; the N sequence variants are mutations at the N ETS transcription factor binding sites; and the wild-type sequence is a non-mutated wild-type sequence of an ETS transcription factor binding site of the N ETS transcription factor binding sites.

15. The method according to any one of claims 1 to 14, wherein the target DNA is a circulating tumor DNA (ctDNA) and the sample is a body fluid sample or the target DNA is tumor DNA and the sample is a tumor sample, the method further comprising:estimating (S5) a tumor mutational burden based on the quantified ctDNA or the tumor DNA.

16. The method according to any one of claims 1 to 13, wherein the target DNA is a target circulating free DNA (cfDNA) and the sample is a body fluid sample.

17. The method according to claim 16, whereinthe target cfDNA is skin-derived cfDNA;the N target portions of the skin-derived cfDNA are E26 transformation-specific (ETS) transcription factor binding sites;the N sequence variants are mutations at the N ETS transcription factor binding sites; and the wild-type sequence is a non-mutated wild-type sequence of an ETS transcription factor binding site of the N ETS transcription factor binding sites.

18. The method according to claim 16, whereinthe target cfDNA is circulating donor DNA;the N target portions of the circulating donor DNA represent N single nucleotide polymorphisms (SNPs) for which a donor and a recipient are both homozygotes but have reciprocal alleles;the N sequence variants are N donor-specific SNP alleles; andthe wild-type sequence is a recipient-specific SNP allele.

19. The method according to claim 16, whereinthe target cfDNA is circulating tumor DNA (ctDNA);the N target portions of the ctDNA are N cancer hotspot genomic portions;the N sequence variants are cancer-inducing mutations at the N cancer hotspot genomic portions; andthe wild-type sequence is a non-muted wild-type sequence of a cancer hotspot genomic portion of the N cancer hotspot genomic portions.

20. The method according to any one of claims 1 to 13, whereinthe target DNA is circulating tumor DNA (ctDNA); andthe N target portions of the ctDNA are N portions that are mutated in the tumor or have a variant sequence that is not a germline variant.

21. The method according to any one of claims 1 to 13, whereinthe sample is a liquid biopsy from a recipient having a transplanted organ from a donor;the target DNA is circulating free DNA (cfDNA); andthe N target portions of the cfDNA are N portions harboring a sequence variation, where both the donor and recipient are homozygotes but with different alleles.

22. The method according to any one of claims 1 to 13, wherein the target DNA is target complementary DNA (cDNA) obtained by reverse transcription of ribonucleic acid (RNA) in a body sample.

23. A method for quantifying a target deoxyribonucleic acid (DNA) in a sample, the method comprising:contacting (S20) the sample with N primer pairs each comprising a forward primer and a reverse primer, N variant padlock probes and a control padlock probe, whereinN is an integer equal to or larger than 2;each primer pair of the N primer pairs is designed to amplify a respective target portion of the target DNA;each variant padlock probe of the N variant padlock probes is complementary to or comprises a sequence variant of a respective target portion of the N target portions;the control padlock probe is complementary to or comprises a wild-type sequence of a target portion of the N target portions;joining (S21) 5’ and 3’ ends of padlock probes of the N variant padlock probes and the control padlock probe while hybridized to the target DNA to form circular variant padlock probes and circular control padlock probes;rolling circle amplifying (S22) the circular variant padlock probes with first labelled amplification primers having a first label and complementary to a portion of the circular variant padlock probes to generate first labelled rolling circle products and the circular control padlock probes with second labelled amplification primers having a second label different from the first label and complementary to a portion of the circular control padlock probes to generate second labelled rolling circle products;detecting (S23) the first labelled rolling circle products and the second labelled rolling circle products; andquantifying (S24) the target DNA based on the detected first and second labelled rolling circle products.

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