Noninvasive methods for assessing transplant rejection in pregnant transplant recipients

Abstract

The present disclosure provides a method for preparing and analyzing a biological sample from a maternal transplant recipient, the method comprising extracting cell-free DNA from the recipient, the cell-free DNA including donor-derived cell-free DNA, recipient-derived cell-free DNA, and fetus-derived cell-free DNA, and measuring the amount of cell-free DNA and donor-derived cell-free DNA to enable assessment of transplant rejection. Detection of donor-derived cell-free DNA can be performed based on loci for which the maternal transplant recipient and the biological father of the fetus are homozygous and the transplant donor is heterozygous.

Description

[Background technology]

[0001] Rapid detection of graft injury and / or rejection remains a challenge for transplant recipients. This challenge is magnified in pregnant mothers, as renal dysfunction and chronic hypertension are common in pregnant kidney transplant recipients. In this population, it can be difficult to distinguish between transplant rejection, progressive kidney disease, and preeclampsia using noninvasive methods.

[0002] Post-transplantation testing to assess and monitor transplant injury and / or rejection is an important aspect of post-transplant care and determining the need for individualized immunosuppressive therapy. In addition to being dangerous to the mother and fetus, conventional biopsy-based testing is invasive, costly, and can lead to delayed diagnosis of transplant injury and / or rejection. Therefore, there is a need for a non-invasive test for transplant injury and / or rejection that is safer, more sensitive, and more specific than conventional biopsy-based testing to enable early diagnosis of transplant injury and / or rejection in pregnant transplant recipients.

[0003] Therefore, improved methods are needed for early and accurate diagnosis, screening, testing, and monitoring of transplant injury and / or rejection in pregnant transplant recipients. The present disclosure addresses this need. Summary of the Invention

[0004] In one aspect, the disclosure relates to a method for preparing a composition of amplified DNA from a biological sample of a maternal transplant recipient useful for determining transplant status, the method comprising: extracting cell-free DNA (cfDNA) from the biological sample of the maternal transplant recipient, wherein the extracted cfDNA includes donor-derived cell-free DNA (dd-cfDNA) from the transplant, recipient-derived cell-free DNA (rd-cfDNA) from the maternal transplant recipient, and fetal-derived cell-free DNA (fd-cfDNA) from the fetus; preparing from the cfDNA a composition enriched for a plurality of target loci, wherein the target loci include one or more SNP loci at which the maternal transplant recipient and the biological father of the fetus are homozygous to ensure fetal homozygosity at the SNP loci such that any heterozygosity observed in the extracted cfDNA at the SNP loci is derived from the transplant; quantifying the amount of dd-cfDNA based on heterozygosity at the SNP loci; and determining whether the amount of dd-cfDNA, or a function thereof, exceeds a cutoff threshold indicative of transplant rejection.

[0005] In some embodiments, quantifying the cfDNA and dd-cfDNA includes preparing a sequencing library from the extracted cfDNA and sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads.

[0006] In some embodiments, the method further comprises sequencing the paternal DNA of the biological father of the fetus, and identifying one or more SNP loci where the maternal transplant recipient and the biological father of the fetus are homozygous, while the transplant dd-cfDNA contains heterozygous alleles. Thus, the amount of sequence reads obtained from the heterozygous alleles of the SNP loci can be used to quantify the amount of dd-cfDNA in the biological sample of the pregnant transplant recipient.

[0007] In some embodiments, the target loci include 10 to 50,000 target loci, or 100 to 20,000 target loci, or 100 to 1,000 target loci, or 1,000 to 10,000 target loci, or 10,000 to 50,000 target loci. In some embodiments, the method further includes enriching the target loci by performing multiplex targeted amplification of DNA at 10 to 50,000 target loci, or 100 to 20,000 target loci, or 100 to 1,000 target loci, or 1,000 to 10,000 target loci, or 10,000 to 50,000 target loci, preferably in a single reaction volume. In some embodiments, the method further comprises enriching the target loci using hybrid capture probes that target 10 to 50,000 target loci, or 100 to 20,000 target loci, or 100 to 1,000 target loci, or 1,000 to 10,000 target loci, or 10,000 to 50,000 target loci, preferably in a single reaction volume.

[0008] In one aspect, the disclosure relates to a method for preparing a composition of DNA derived from a biological sample of a maternal transplant recipient, useful for assessing transplant rejection, wherein the composition of DNA comprises one or more target loci comprising transplant-derived alleles, and the assessing step further comprises determining the amount of the transplant-derived allele at one or more SNP loci and determining whether the amount of the transplant-derived allele at the one or more SNP loci, or a function thereof, exceeds a cutoff threshold indicative of transplant rejection, wherein transplant rejection is assessed by a combination of (i) the amount of the transplant-derived allele at the one or more target loci, or a function thereof, and (ii) the total amount of dd-cfDNA or a percentage of dd-cfDNA.

[0009] In one aspect, the present disclosure relates to a method of administering immunosuppressive therapy in a maternal transplant recipient, the method comprising: (a) quantifying the total amount of cfDNA and the amount of dd-cfDNA in a biological sample from the transplant recipient according to a method described herein; and (b) titrating the dosage of the immunosuppressive therapy according to the amount of cfDNA, or a function thereof, and the amount of dd-cfDNA, or a function thereof. In some embodiments, the method further comprises repeating step (a) longitudinally for the same transplant recipient and determining longitudinal changes in the amount of cfDNA, or a function thereof, and the amount of dd-cfDNA, or a function thereof. In some embodiments, the method further comprises titrating the dosage of the immunosuppressive therapy according to the longitudinal changes in the total amount of cfDNA, or a function thereof, and the amount of dd-cfDNA, or a function thereof.

[0010] In some embodiments of the methods described herein, an increase in the level of dd-cfDNA indicates transplant rejection and the need for adjustment of immunosuppressive therapy. In some embodiments of the methods described herein, a change or decrease in the level of dd-cfDNA indicates transplant tolerance or stability and the need for adjustment of immunosuppressive therapy.

[0011] In some embodiments of the methods described herein, the selected target locus comprises one or more single nucleotide polymorphisms (SNPs).

[0012] In some embodiments, the methods described herein are performed without prior knowledge of the donor and / or recipient genotype.

[0013] In some embodiments of the methods described herein, universal amplification of the extracted DNA is performed. In some embodiments, the universal amplification preferentially amplifies dd-cfDNA over rd-cfDNA and fd-cfDNA.

[0014] In some embodiments of the methods described herein, extracting cfDNA from the biological sample includes size selection to enrich for dd-cfDNA and reduce the amount of rd-cfDNA and fd-cfDNA.

[0015] In some embodiments of the methods described herein, the amount of cfDNA is measured by quantitative PCR, real-time PCR, digital PCR, sequencing, microarray, or molecular barcoding and microscopic imaging (such as NanoString nCounter®). In some embodiments of the methods described herein, the amount of dd-cfDNA is determined using ratiometric and / or machine learning artificial intelligence comparisons at single or multiple time points.

[0016] In some embodiments of the methods described herein, the cutoff threshold is an estimated percentage of dd-cfDNA relative to total cfDNA or a function thereof. In some embodiments of the methods described herein, an amount of dd-cfDNA greater than 1% of total cfDNA indicates that the transplant will undergo acute rejection, and an amount of dd-cfDNA less than 1% of total cfDNA indicates that the transplant will undergo borderline rejection, other damage, or is stable.

[0017] In some embodiments of the methods described herein, the transplant recipient has received one or more transplants selected from kidney, liver, pancreas, intestine, heart, lung, heart / lung, stomach, testis, penis, ovary, uterus, thymus, face, hand, leg, bone, bone marrow, cornea, skin, pancreatic islet cells, heart valve, blood vessel, and blood transfusion. In some embodiments of the methods described herein, the transplant recipient is obtained within 18 months of transplantation.

[0018] In some embodiments of the methods described herein, the rejection risk of the transplant recipient is determined using logistic regression, random forest, or decision tree machine learning analysis. In some embodiments of the methods described herein, the logistic regression, random forest, or decision tree machine learning analysis further incorporates one or more parameters selected from time since transplant, age of the transplant recipient and / or transplant donor, and gender of the transplant recipient and / or transplant donor.

[0019] In some embodiments of the methods described herein, the biological sample is blood, serum, plasma, or urine. [Brief explanation of the drawings]

[0020] [Figure 1] Workflow for detecting donor-derived cell-free DNA in human plasma samples from pregnant transplant recipients. [Figure 2] Workflow for measuring dd-cfDNA percentage in pregnant kidney transplant recipients (KTRs). [Figure 3] Vignettes showing dd-cfDNA testing and medical history in two pregnant KTRs. [Figure 4] Heterozygosity plots in two pregnant KTRs with (a) low DFE and (b) high DFE. [Figure 5] This is a measure of the donor-derived proportion of pregnant people with KTR. DETAILED DESCRIPTION OF THE INVENTION

[0021] Sigdel et al., “Optimizing Detection of Kidney Transplant Injury by Assessment of Donor-Derived Cell-Free DNA via Massively Multiplex PCR,” J. Clin. Med. 8(1):19 (2019), is incorporated herein by reference in its entirety.

[0022] WO2020 / 010255, filed as PCT / US2019 / 040603 on July 3, 2019, and entitled "Methods for Detection of Donor-Derived Cell-Free DNA," is hereby incorporated by reference in its entirety.

[0023] WO2021 / 243045, filed as PCT / US2021 / 034561 on May 27, 2021, and entitled "Methods for Detection of Donor-Derived Cell-Free DNA," is hereby incorporated by reference in its entirety.

[0024] The present disclosure relates to methods for determining and monitoring transplant rejection in pregnant recipients based on targeted enrichment and high-throughput sequencing of cell-free DNA from biological samples of the transplant recipient. In some embodiments, cell-free DNA (cfDNA) is isolated from a biological sample, such as a blood, plasma, serum, or urine sample, of the transplant recipient. In some embodiments, the examples provided herein demonstrate that the presently disclosed methods can be used to detect donor-derived nucleic acids in biological samples collected from pregnant transplant recipients.

[0025] A method for determining and monitoring transplant rejection based on the measurement of cell-free DNA. In one aspect, the disclosure provides a method for preparing a composition of DNA from a biological sample of a maternal transplant recipient useful for determining transplant status, comprising: (a) extracting cell-free DNA (cfDNA) from a biological sample of a maternal transplant recipient, wherein the extracted cfDNA includes donor-derived cell-free DNA (dd-cfDNA) from the transplant, recipient-derived cell-free DNA (rd-cfDNA) from the maternal transplant recipient, and fetal-derived cell-free DNA (fd-cfDNA) from the fetus; (b) preparing a composition enriched for one or more selected target loci from cfDNA, wherein the maternal transplant recipient and the biological father of the fetus are homozygous at the selected target loci to ensure homozygosity of the fetus at the selected target loci such that heterozygosity observed in the extracted cfDNA at the selected target loci is derived from the transplant; and (c) quantifying the amount of extracted cfDNA and the amount of dd-cfDNA based on heterozygosity at the selected target loci and determining whether the amount of dd-cfDNA or a function thereof exceeds a cutoff threshold indicative of transplant rejection. In some embodiments, the method includes preparing a sequencing library from the extracted cfDNA and sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads. In some embodiments, no amplification or pre-amplification is performed on the extracted cfDNA prior to sequencing. In some embodiments, the selected loci include 10 to 50,000 target loci, and the method further includes performing multiplexed target amplification of DNA at 10 to 50,000 target loci in a single reaction volume.

[0026] In one aspect, the disclosure provides a method for preparing a composition of DNA from a biological sample of a maternal transplant recipient useful for determining transplant status, comprising: (a) extracting cfDNA from a biological sample of a maternal transplant recipient, wherein the extracted cfDNA includes donor-derived cell-free DNA (dd-cfDNA), recipient-derived cell-free DNA (rd-cfDNA), and fetal-derived cell-free DNA (fd-cfDNA); (b) preparing a sequencing library from the extracted cfDNA, sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads, and quantifying the total amount of dd-cfDNA based on the sequencing reads; and (c) determining whether the amount of dd-cfDNA or a function thereof exceeds a cutoff threshold indicative of transplant rejection or damage. In some embodiments, no amplification or pre-amplification is performed on the extracted cfDNA prior to sequencing. In some embodiments, multiplex target amplification is performed on the extracted cfDNA at 10 to 50,000 target loci in a single reaction volume prior to sequencing. In some embodiments, preparing the sequencing library includes attaching adapters to the extracted cfDNA, for example, by ligation. In some embodiments, attaching adapters to the extracted cfDNA includes end repair, adding adenosines to the cfDNA fragments, followed by cohesive end ligation to the cfDNA fragments. In some embodiments, the cfDNA fragments are repaired and the generated blunt ends are filled in. In some embodiments, attaching adapters to the extracted cfDNA includes blunt end ligation of adapters to the cfDNA fragments. In some embodiments, the adapters are attached during the amplification step.

[0027] In one aspect, the disclosure provides a method for preparing a composition of amplified DNA from a biological sample of a maternal transplant recipient useful for determining transplant status, comprising: (a) extracting cfDNA from a biological sample of a maternal transplant recipient, wherein the extracted cfDNA includes dd-cfDNA, rd-cfDNA, and cell-free DNA from the transplant and derived from the fetus; (b) preparing a composition of amplified DNA by performing multiplex target amplification of the extracted cfDNA at 10 to 50,000 target loci in a single reaction volume to detect and quantify the amount of dd-cfDNA; (c) determining whether the amount of dd-cfDNA or a function thereof exceeds a cutoff threshold indicative of transplant rejection.

[0028] In one aspect, the disclosure provides a method for preparing a composition of amplified DNA from a biological sample of a maternal transplant recipient useful for determining transplant status, comprising: (a) extracting cfDNA from the blood of the maternal transplant recipient, wherein the extracted cfDNA includes dd-cfDNA, rd-cfDNA, and fd-cfDNA; (b) preparing a composition of amplified DNA by performing targeted amplification of the extracted cfDNA at 10 to 50,000 target loci in a single reaction volume; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of dd-cfDNA, transplant rd-cfDNA, and fd-cfDNA based on the sequencing reads, wherein the loci of the donor, recipient, and fetus are the same, and the reads of the donor, recipient, and fetus are distinguished based on the insertion sequence; (c) determining whether the percentage of dd-cfDNA or a function thereof exceeds a cutoff threshold indicative of transplant rejection. As used herein, "insertion sequence" refers to any sequence that differs at a target locus in a transplant recipient compared to the same target locus in a transplant donor.

[0029] In one aspect, the disclosure provides a method for preparing a composition of amplified DNA from a biological sample of a maternal transplant recipient useful for determining transplant status, comprising: (a) extracting cfDNA from a biological sample of a maternal transplant recipient, wherein the extracted cfDNA includes dd-cfDNA, rd-cfDNA, and fd-cfDNA; (b) preparing a composition of amplified DNA by performing targeted amplification of the extracted cfDNA at 10 to 50,000 target loci in a single reaction volume, wherein the target loci include one or more target loci indicative of transplant rejection; (c) determining the amount of one or more target loci indicative of transplant rejection and determining whether the amount of, or a function thereof, of the one or more target loci indicative of transplant rejection exceeds a cutoff threshold indicative of transplant rejection.

[0030] In one aspect, the disclosure provides a method for preparing a composition of DNA derived from a biological sample of a maternal transplant recipient, useful for determining transplant status, wherein the composition of DNA comprises one or more target loci indicative of transplant rejection, and the determining step further comprises determining the amount of the one or more target loci indicative of transplant rejection and determining whether the amount or function thereof of the one or more target loci indicative of transplant rejection exceeds a cutoff threshold indicative of transplant rejection, wherein transplant rejection is determined by a combination of (i) the amount or function thereof of the one or more target loci indicative of transplant rejection and (ii) the total amount of dd-cfDNA or the percentage of dd-cfDNA.

[0031] In one aspect, the present disclosure provides a method of administering immunosuppressive therapy in a maternal transplant recipient, comprising: (a) quantifying the amount of total cfDNA and dd-cfDNA in a biological sample of a transplant recipient according to any of the methods disclosed herein; (b) titrating the dosage of immunosuppressive therapy according to the amount of, or a function thereof, of cfDNA and the amount of, or a function thereof, of dd-cfDNA. In some embodiments, the method further comprises repeating step (a) longitudinally in the same transplant recipient and determining longitudinal changes in the amount of, or a function thereof, of cfDNA and longitudinal changes in the amount of, or a function thereof, of dd-cfDNA.

[0032] In some embodiments, the cfDNA is derived from extracellular vesicles (EVs) isolated from a biological sample, such as a blood, plasma, serum, or urine sample, of a maternal transplant recipient.

[0033] Samples containing nucleic acids and methods for obtaining samples and extracting nucleic acids - Patents.com The methods disclosed herein include extracting fragmented or intact cfDNA from a sample obtained from a maternal transplant recipient. In some embodiments, the transplant recipient is a human subject and the transplant donor is a human. In some embodiments, the transplant is from a pig, a primate, a baboon, a cow, or a dog. In some embodiments, the transplant may be an allograft or a xenograft. In some embodiments, the transplant may be an organ transplant, a tissue transplant, a cell transplant, or a fluid transplant.

[0034] In some embodiments, the transplant recipient receives multiple transplanted organs selected from kidney, liver, pancreas, intestine, heart, lung, heart / lung, stomach, testis, penis, ovary, uterus, thymus, face, hand, leg, bone, bone marrow, cornea, skin, pancreatic islet cells, heart valve, and blood vessel. In some embodiments, one or more transplanted organs are from the same transplant donor. In some embodiments, one or more transplanted organs are from more than one different transplant donor. In some embodiments, the transplant recipient has received multiple simultaneous organ transplants. In some embodiments, the transplant recipient has received a blood transfusion. In some embodiments, the blood transfusion is from the same donor as one or more of the organ transplants. In some embodiments, the blood transfusion is from a different donor to the organ donor.

[0035] In some embodiments, the transplant recipient has received one or more transplanted organs selected from kidney, liver, heart, lung, pancreas, intestine, thymus, and uterus. In some embodiments, the transplant recipient has received a kidney transplant. In some embodiments, the transplant recipient has received a liver transplant. In some embodiments, the transplant recipient has received a heart transplant. In some embodiments, the transplant recipient has received a lung transplant. In some embodiments, the transplant recipient has received a pancreas transplant. In some embodiments, the transplant recipient has received an intestine transplant. In some embodiments, the transplant recipient has received a thymus transplant. In some embodiments, the transplant recipient has received a uterus transplant.

[0036] In some embodiments, the sample is obtained from a transplant recipient less than 18 months post-transplant, less than 17 months post-transplant, less than 16 months post-transplant, less than 15 months post-transplant, less than 14 months post-transplant, less than 13 months post-transplant, or less than 12 months post-transplant, hi some embodiments, the sample is obtained from a transplant recipient between 0-2 months post-transplant, 2-4 months post-transplant, 4-6 months post-transplant, 6-9 months post-transplant, 9-12 months post-transplant, or 12-18 months post-transplant.

[0037] In some embodiments, the transplant recipient is pregnant. In some embodiments, the transplant recipient is in the first trimester of pregnancy. In some embodiments, the transplant recipient is in the second trimester of pregnancy. In some embodiments, the transplant recipient is in the third trimester of pregnancy. In some embodiments, the transplant recipient is less than three months pregnant. In some embodiments, the transplant recipient is less than six months pregnant. In some embodiments, the transplant recipient is less than nine months pregnant. In some embodiments, the transplant recipient is nine months or more pregnant. In some embodiments, the transplant recipient has recently given birth. In some embodiments, the transplant recipient is less than one day postpartum, i.e., less than one day postpartum. In some embodiments, the transplant recipient is less than one week postpartum, i.e., less than one week postpartum. In some embodiments, the transplant recipient is less than one month postpartum, i.e., less than one month postpartum. In some embodiments, the transplant recipient is less than three months postpartum, i.e., less than three months postpartum. In some embodiments, the transplant recipient is less than one year postpartum, i.e., less than one year postpartum. In some embodiments, there is a single fetus. In some embodiments, there are multiple fetuses. In some embodiments, the transplant recipient is not pregnant.

[0038] In some embodiments, the methods disclosed herein further comprise longitudinally measuring the amounts of total cfDNA and dd-cfDNA in the same transplant recipient and determining longitudinal changes in the amounts of total cfDNA and dd-cfDNA. In some embodiments, the amount of dd-cfDNA is the total amount of cfDNA derived from the donor organ.

[0039] In some embodiments, the transplant recipient has received one or more organs from the same transplant donor. In some embodiments, the transplant recipient has received one or more organs from multiple different transplant donors. In some embodiments, the transplant recipient has received multiple simultaneous organ transplants from one or more different donors.

[0040] The biological sample may be a bodily fluid sample, tissue, organ, or individual cells. In some embodiments, the biological sample comprises blood, plasma, serum, CSF, or urine. In some embodiments, the biological sample is blood. In some embodiments, the sample is blood, plasma, or serum. In some embodiments, the biological sample may be extracellular vesicles derived from a bodily fluid sample such as blood, plasma, serum, CSF, or urine.

[0041] Nucleic acids and methods for extracting or concentrating nucleic acids The methods disclosed herein include extracting nucleic acids from a sample derived from a subject. In some embodiments, the subject may be a pregnant transplant recipient. In some embodiments, the subject may be the father of a fetus in a transplant recipient. In some embodiments, the subject may be a transplant donor. In some embodiments, the subject may be a fetus. Nucleic acid may refer to genomic DNA, cDNA, cell-free DNA (cfDNA), cell-free mitochondrial DNA (cfmDNA), cell-free DNA derived from nuclear DNA (cfnDNA), cellular DNA, or mitochondrial DNA. cfDNA may be derived from exosomes or microvesicles. In some embodiments, nucleic acid may be RNA, such as cell-free RNA, cellular RNA, or RNA extracted from exosomes. The term "RNA" as used herein refers to any type of RNA, including messenger RNA (mRNA) or small non-coding RNA (sncRNA), such as microRNA (miRNA), or a mixture thereof. In some embodiments, the RNA may be cell-free, cellular, or exosomal RNA. In some embodiments, the RNA includes small non-coding RNA (sncRNA). In some embodiments, the sncRNA comprises a microRNA (miRNA), a piwi-interacting RNA (piRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), or other RNA (miscRNA). In some embodiments, the cell-free sncRNA is derived from exosomes or microvesicles.

[0042] In some embodiments, nucleic acids are extracted by size exclusion. In some embodiments, cfDNA is separated from cellular DNA based on size. In some embodiments, nucleic acids are isolated by using magnetically or otherwise labeled affinity chromatography.

[0043] In some embodiments, nucleic acids are preferentially enriched. Nucleic acids may be preferentially enriched by using preferential enrichment at loci or target sites. Such preferential enrichment refers to any method whereby the proportion of nucleic acid molecules corresponding to loci in a nucleic acid mixture after enrichment is higher than the proportion of nucleic acid molecules corresponding to loci in the nucleic acid mixture before enrichment. The method may include selective amplification of nucleic acid molecules corresponding to loci. The method may include removing nucleic acid molecules that do not correspond to loci. The method may include a combination of methods. Enrichment is defined as the proportion of nucleic acid molecules corresponding to loci or targets in the mixture after enrichment divided by the proportion of nucleic acid molecules corresponding to loci or targets in the mixture before enrichment. Preferential enrichment may be performed at multiple loci. In some embodiments of the present disclosure, the enrichment is greater than 20. In some embodiments of the present disclosure, the enrichment is greater than 200. In some embodiments of the present disclosure, the enrichment is greater than 2,000. When preferential enrichment is performed at multiple loci, the enrichment may refer to the average enrichment of all loci in the set of loci.

[0044] Amplification refers to techniques that increase the copy number of a nucleic acid molecule. Selective amplification can refer to techniques that increase the copy number of a specific nucleic acid molecule or a nucleic acid molecule corresponding to a specific region of a nucleic acid molecule. It can also refer to methods that increase the copy number of a specific target molecule or target region of a nucleic acid molecule over the copy number of non-target molecules or regions of a nucleic acid molecule.

[0045] Selective amplification can be a preferential enrichment method. A universal priming sequence refers to a DNA sequence that can be added to a population of target DNA molecules, for example, by ligation, PCR, or ligation-mediated PCR. Once added to a population of target molecules, primers specific to the universal priming sequence can be used to amplify the target population using a single amplification primer pair. The universal priming sequence is typically not related to the target sequence. A universal adapter, or ligation adapter or library tag, is a DNA molecule that contains a universal priming sequence that can be covalently attached to the 5' and 3' ends of a population of target double-stranded DNA molecules. Addition of the adapter provides universal priming sequences at the 5' and 3' ends of the target population, where PCR amplification can be performed, and a single amplification primer pair is used to amplify all molecules from the target population. Targeting refers to a method used to selectively amplify or otherwise preferentially enrich molecules of DNA corresponding to a set of loci in a mixture of DNA.

[0046] Specific nucleic acids may also be enriched using hybrid capture. In some embodiments, preferentially enriching DNA at multiple loci or target sites includes obtaining a set of hybrid capture probes, hybridizing the hybrid capture probes to DNA in a sample, and physically separating the hybridized DNA from unhybridized DNA from the sample of DNA.

[0047] In some embodiments, in the methods disclosed herein, DNA is preferentially enriched at target loci or biomarkers.

[0048] The term "biomarker" refers to a molecule that is an indicator of an abnormal biological state (e.g., a disease or disorder, or transplant rejection). For example, a biomarker can be (a) expressed at a higher or lower level, (b) have an altered ratio compared to another biomarker, (c) present at a higher or lower level, (d) a mutation or variant of a gene product, or (e) simply a gene or gene product (i.e., RNA or protein) that is present or absent in a cell or tissue sample from a subject with or suspected of having a disease, compared to a non-diseased tissue or cell sample from a subject with or suspected of having a disease, or compared to a cell or tissue sample from a subject or pool of subjects without or not suspected of having a disease. In the context of transplantation, a biomarker can be indicative of poor donor organ health or transplant rejection. That is, one or more gene products are sufficiently specific to the test sample that one or more can be used to identify, predict, or detect the presence of transplant rejection, disease, disease risk, risk of a predetermined event, or change in disease state, or to inform appropriate or improved treatment regimens. In the methods described herein, levels of dd-cfDNA above a threshold may be considered a biomarker.

[0049] In some embodiments, one or more biomarkers are a genetic abnormality or a set of genetic abnormalities, which are used herein to refer to the amount and variants of nucleic acids in nucleic acid-containing particles. Specifically, genetic abnormalities include, but are not limited to, overexpression of a gene (e.g., an oncogene) or a panel of genes, underexpression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or a panel of genes, alternative splice variants of a gene or a panel of genes, gene copy number variations (CNVs) (e.g., DNA double minute chromosomes), nucleic acid modifications (e.g., methylation, acetylation, and phosphorylation), single nucleotide polymorphisms (SNPs), chromosomal rearrangements (e.g., inversions, deletions, and duplications), mutations (insertion, deletion, duplication, missense, nonsense, synonymous, or any other nucleotide changes) of a gene or a panel of genes (which often ultimately affect the activity and function of the gene product, leading to alternative transcriptional splice variants and / or altered gene expression levels), or any combination of the above.

[0050] In some embodiments, preferentially enriching DNA in a sample at a plurality of polymorphic loci includes obtaining a plurality of pre-circularized probes, each probe targeting one of the polymorphic loci and the 3' and 5' ends of the probes designed to hybridize to a region of DNA separated from the polymorphic site at the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the pre-circularized probes to DNA from the sample; using a DNA polymerase to fill in the gaps between the ends of the hybridized probes; circularizing the pre-circularized probes; and amplifying the circularized probes.

[0051] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci includes obtaining a plurality of ligation-mediated PCR probes, each PCR probe targeting one of the polymorphic loci and wherein the upstream and downstream PCR probes are designed to hybridize to a DNA region on one strand of the DNA separated from the polymorphic site of the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the ligation-mediated PCR probes to DNA from the first sample; using a DNA polymerase to fill gaps between the ends of the ligation-mediated PCR probes; ligating the ligation-mediated PCR probes; and amplifying the ligated ligation-mediated PCR probes.

[0052] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci includes obtaining a plurality of hybrid capture probes that target the polymorphic loci, hybridizing the hybrid capture probes to DNA in the sample, and physically removing some or all of the unhybridized DNA from the first DNA sample.

[0053] In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping. In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping, and the length of the capture probes on either side can be selected from the group consisting of less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases. In some embodiments, the hybrid capture probes are designed to hybridize to regions that overlap the polymorphic site, and the plurality of hybrid capture probes includes at least two hybrid capture probes for each polymorphic locus, each hybrid capture probe designed to be complementary to a different allele at the polymorphic locus.

[0054] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci includes obtaining a plurality of inner forward primers, each primer targeting one of the polymorphic loci, the 3' end of the inner forward primer designed to hybridize to a DNA region upstream of the polymorphic site and separated from the polymorphic site by a small number of bases, wherein the small number of bases is selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, or 31-60 base pairs; and optionally, each primer targeting a region of the polymorphic locus. The method includes obtaining a plurality of inner reverse primers that target one of the loci, the inner reverse primers being designed such that the 3' ends of the inner reverse primers hybridize to a DNA region upstream of the polymorphic site and are separated from the polymorphic site by a small number of bases, where the small number of bases is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, or 31 to 60 base pairs; hybridizing the inner primers to the DNA; and amplifying the DNA using polymerase chain reaction to form an amplicon.

[0055] In some embodiments, the method also includes obtaining a plurality of outer forward primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA upstream of the inner forward primer; optionally obtaining a plurality of outer reverse primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA immediately downstream of the inner reverse primer; hybridizing the first primer to the DNA; and amplifying the DNA using polymerase chain reaction.

[0056] In some embodiments, the method also includes obtaining a plurality of outer reverse primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA immediately downstream of the inner reverse primer; optionally obtaining a plurality of outer forward primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA upstream of the inner forward primer; hybridizing the first primer to the DNA; and amplifying the DNA using polymerase chain reaction.

[0057] In some embodiments, preparing the sample includes incorporating universal adapters into DNA and amplifying the DNA using polymerase chain reaction, wherein at least a portion of the amplified amplicons are less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp, and wherein the portion is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%.

[0058] In some embodiments, amplifying the DNA is carried out in one or more separate reaction volumes, and each separate reaction volume contains more than 10 different forward and reverse primer pairs, more than 100 different forward and reverse primer pairs, more than 200 different forward and reverse primer pairs, more than 500 different forward and reverse primer pairs, more than 1,000 different forward and reverse primer pairs, more than 2,000 different forward and reverse primer pairs, more than 5,000 different forward and reverse primer pairs, more than 10,000 different forward and reverse primer pairs, more than 20,000 different forward and reverse primer pairs, more than 50,000 different forward and reverse primer pairs, or more than 100,000 different forward and reverse primer pairs.

[0059] In some embodiments, preparing the sample further comprises dividing the sample into multiple portions, with the DNA in each portion preferentially enriched for a subset of the multiple polymorphic loci. In some embodiments, the inner primers are selected by identifying primer pairs that are likely to form undesired primer duplexes and removing at least one of the identified primer pairs that are likely to form undesired primer duplexes from the multiple primers. In some embodiments, the inner primers include a region designed to hybridize either upstream or downstream of the targeted polymorphic locus, and optionally include a universal priming sequence designed to enable PCR amplification. In some embodiments, at least some of the primers further include a random region that varies for each individual primer molecule. In some embodiments, at least some of the primers further include a molecular barcode.

[0060] In some embodiments, the method includes (a) performing a multiplex polymerase chain reaction (PCR) on a nucleic acid sample containing target loci to simultaneously amplify at least 10 different target loci in a single reaction volume using (i) at least 10 different primer pairs, or (ii) at least 10 target-specific primers and either a universal primer or a tag-specific primer, to generate amplification products containing target amplicons, and (b) sequencing the amplified products. In some embodiments, the method does not include using a microarray.

[0061] In some embodiments, the method includes: (a) performing a multiplex polymerase chain reaction (PCR) on a cfDNA sample containing target loci to simultaneously amplify at least 10 different target loci in a single reaction volume using (i) at least 10 different primer pairs, or (ii) at least 10 target-specific primers and either a universal primer or a tag-specific primer, to generate amplification products containing target amplicons; and (b) sequencing the amplified products. In some embodiments, the method does not include using a microarray.

[0062] After blood collection and before nucleic acid extraction, blood cells in the blood sample may burst and release long DNA fragments into the sample, thereby increasing the total amount of cfDNA and background noise, distorting the percentage of detected dd-cfDNA. To reduce such background noise, two specific enrichment methods for dd-cfDNA have been considered, based on the observation that dd-cfDNA is usually shorter than the DNA shredded from blood cells of transplant recipients. In one embodiment, size selection is applied to select shorter cfDNA. In another embodiment, a universal amplification step is applied to reduce noise (e.g., before applying multiplex PCR), based on the hypothesis that short dd-cfDNA (often in mononucleosomal form) is amplified more efficiently than long DNA from transplant recipients.

[0063] Target Genes and Loci In some embodiments, the method disclosed herein comprises selecting the target locus that is homozygous in both maternal transplant recipient and the biological father of fetus, and ensures the homozygosity of fetus.Therefore, any heterozygosity at the locus detected in extracted cfDNA is derived from donor.In some embodiments, the heterozygosity detected in extracted cfDNA can be used to quantify the amount of dd-cfDNA present in biological sample.

[0064] In some embodiments, the methods disclosed herein further comprise preferentially enriching the cfDNA at multiple target loci selected based on the homozygosity of both the maternal transplant recipient and the biological father of the fetus. In some embodiments, between 10 and 50,000 target loci are selected and enriched.

[0065] In some embodiments, the methods described herein further comprise sequencing the paternal genome of the fetus to select target loci for which both the maternal transplant recipient and the biological father of the fetus are homozygous. In some embodiments, the paternal genotype of the fetus is already known.

[0066] The nucleic acids may include biomarkers indicative of an immune response or various diseases or conditions described elsewhere herein. In some embodiments, the target loci include one or more different sets of target loci. In some embodiments, the target loci include a set of recipient target loci, a set of fetal target loci, and a set of donor target loci, where each set of target loci in the recipient, fetus, and donor is different. In some embodiments, the sets of target loci in the recipient and fetus are the same, and the sets of donor target loci are different. In some embodiments, each set of target loci in the recipient, fetus, and donor are all the same, where one or more recipient and / or fetal target loci can be distinguished from the corresponding donor locus by an insertion sequence. As used herein, "insertion sequence" refers to any sequence in which a target locus in a transplant recipient and / or fetus is different from the same target locus in the transplant donor.

[0067] In some embodiments, the method includes extracting fragmented or intact cfDNA from a sample of a transplant recipient, wherein the extracted cfDNA includes cfDNA from the donor, fetus, and / or recipient, and the cfDNA includes a plurality of biomarkers indicative of an immune response or a disease or disorder. In some embodiments, the biomarkers indicate an increased immune response. In some embodiments, the biomarkers indicate a decreased immune response.

[0068] In some embodiments, the presently disclosed method includes preselecting a cfDNA target molecule. In some embodiments, the cfDNA target molecule includes a cfDNA species known to be associated with organ health assessment. In some embodiments, the present disclosure provides a method for identifying a cfDNA target molecule associated with organ health assessment.

[0069] In some embodiments, the methods disclosed herein further comprise preferentially enriching the cfDNA at multiple target loci or biomarkers indicative of transplant rejection. In some embodiments, the cfDNA biomarkers are indicative of an increased immune response or a decreased immune response.

[0070] In some embodiments, the target loci and / or biomarkers comprise single nucleotide polymorphism (SNP) loci.

[0071] Samples and methods for isolating nucleic acids from samples In some embodiments, the nucleic acid sample includes fragmented or digested nucleic acids. In some embodiments, the nucleic acid sample includes DNA, such as genomic DNA, cDNA, cell-free DNA (cfDNA), cell-free mitochondrial DNA (cf mDNA), cell-free DNA derived from nuclear DNA (cf nDNA), cellular DNA, or mitochondrial DNA.

[0072] In some embodiments, the nucleic acid sample contains DNA from a single cell, 2 cells, 3 cells, 4 cells, 5 cells, 6 cells, 7 cells, 8 cells, 9 cells, 10 cells, or more than 10 cells. In some embodiments, the nucleic acid sample is a substantially cell-free blood or plasma sample. In some embodiments, the nucleic acid sample comprises or is derived from blood, plasma, saliva, semen, sperm, cell culture supernatant, mucus secretions, dental plaque, gastrointestinal tissue, stool, urine, hair, bone, body fluids, tears, tissue, skin, nails, germ cells, embryos, amniotic fluid, chorionic villus samples, bile, lymph, cervical mucus, or a forensic sample. In some embodiments, the target locus is a fragment of nucleic acid. In some embodiments, the target locus is a fragment of nucleic acid found in a genome. In some embodiments, the target locus comprises or consists of a single nucleotide polymorphism (SNP). In some embodiments, the primer is a DNA molecule.

[0073] In some embodiments, the method includes isolating or purifying DNA. There are several standard procedures known in the art to achieve this goal. In some embodiments, the sample may be centrifuged to separate the various layers. In some embodiments, DNA may be isolated using filtration. In some embodiments, DNA preparation may include amplification, separation, chromatographic purification, liquid separation, isolation, preferential enrichment, preferential amplification, target amplification, or any of several other techniques known in the art or described herein. In some embodiments for DNA isolation, RNase is used to degrade RNA. In some embodiments, a QIAamp™ DNA Mini Kit (Qiagen) is used to isolate DNA according to the manufacturer's protocol. In some embodiments, cfDNA molecules are isolated using a MagMAX™ Cell-Free DNA Isolation Kit (Applied Biosystems). DNA concentration and purity may optionally be determined using Nanovue (GE Healthcare, Piscataway, NJ, USA), and DNA integrity may optionally be measured using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

[0074] In some embodiments, adapters are added to generate a sequencing library. Prior to ligation, the sample DNA may be blunt-ended, and then a single adenosine base is added to the 3' end. In some embodiments, ligation of the adapter to the nucleic acid is cohesive end ligation. Prior to ligation, the DNA may be cleaved using a restriction enzyme or some other cleavage method. During ligation, the 3'-terminal adenosine of the sample fragment and the complementary 3'-terminal tyrosine overhang of the adapter can increase ligation efficiency. In some embodiments, adapter ligation is performed using a ligation kit found in an AGILENT SURESELECT™ kit. In some embodiments, adapters are incorporated into the sequence by PCR. In some embodiments, adapters are incorporated into the sequence during universal amplification or targeted amplification.

[0075] In some embodiments, the library is amplified using universal primers. In one embodiment, the amplified library is fractionated by size separation or by using products such as AGENCOURT AMPURE™ beads or other similar methods. In some embodiments, PCR amplification is used to amplify the target loci. In some embodiments, the amplified DNA is sequenced (such as by sequencing using an ILLUMINA IIGAX™ or HiSeq sequencer). In some embodiments, the amplified DNA is sequenced from each end of the amplified DNA to reduce sequencing errors. If a sequence error exists at a particular base when sequencing from one end of the amplified DNA, there is less likely to be a sequence error in the complementary base when sequencing from the other end of the amplified DNA (compared to multiple sequencing from the same end of the amplified DNA).

[0076] As non-limiting examples, the locus may be a single nucleotide polymorphism (SNP), an intron, or an exon. In some embodiments, the locus may include an insertion, deletion, or rearrangement. In some embodiments, the sample may include a blood, serum, or plasma sample. In some embodiments, the sample may include free-floating DNA (e.g., circulating cell-free tumor DNA, circulating self-reader DNA, or circulating cell-free fetal DNA) in a blood, serum, or plasma sample. In these embodiments, the sample is typically from an animal, such as a mammal or human, and is typically present in fragments of approximately 160 nucleotides in length. In some embodiments, free-floating DNA is isolated from blood using EDTA-2Na tubes after centrifugation to remove cellular debris and platelets. Plasma samples can be stored at -80°C until DNA is extracted using, for example, a QIAamp™ DNA Mini Kit (Qiagen, Hilden, Germany) (e.g., Hamakawa et al., Br J Cancer. 2015, 112:352-356).

[0077] Many kits and methods for generating libraries of nucleic acid molecules for subsequent sequencing are known in the art. Kits specifically adapted for preparing libraries from small nucleic acid fragments, particularly circulating cell-free DNA, can be useful for performing the methods provided herein. For example, the NEXTflex™ Cell Free Kit (Bioo Scientific, Austin, Texas) or the Natera Library Prep Kit (Natera, San Carlos, California). Such kits are typically modified to include adapters customized for the amplification and sequencing steps of the methods provided herein. Adapter ligation can also be performed using commercially available kits, such as the ligation kit found in the Agilent SureSelect™ Kit (Agilent, Santa Clara, California).

[0078] The sample nucleic acid molecule is composed of natural or unnatural ribonucleotides or deoxyribonucleotides linked via phosphodiester bonds. Furthermore, the sample nucleic acid molecule is composed of nucleic acid fragments targeted for sequencing. The sample nucleic acid molecule can be or contain nucleic acid fragments at least 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides in length. In any of the embodiments disclosed herein, the sample nucleic acid molecules or nucleic acid fragments may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the lower end of the range and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the upper end of the range. The length can be between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides. In some embodiments, the nucleic acid molecule can be a fragment of genomic DNA and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the lower end of the range and 1 at the upper end of the range. The nucleic acid may be between 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length. For clarity, nucleic acids initially isolated from biological tissues, fluids, or cultured cells may be much longer than the sample nucleic acid molecules processed using the methods herein.As discussed herein, for example, such initially isolated nucleic acid molecules can be fragmented to generate nucleic acid fragments before use in the methods herein. In some embodiments, the nucleic acid molecule and the nucleic acid fragment can be identical. The sample nucleic acid molecule or sample nucleic acid fragment can include a target locus that contains the nucleotide(s) being queried, particularly a single nucleotide polymorphism or single nucleotide mutation. In any of the disclosed embodiments, the target locus can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides in length and can include a portion or the entire sample nucleic acid molecule and / or sample nucleic acid fragment. In other embodiments, the target locus is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the lower end of the range and 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length. In some embodiments, the target loci of different sample nucleic acid molecules can be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical. In some embodiments, target loci of different sample nucleic acid molecules can share at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity.

[0079] In some embodiments, the entire sample nucleic acid molecule is a sample nucleic acid fragment. For example, in certain embodiments where an adapter is ligated directly to the end of the sample nucleic acid molecule, or to a nucleic acid(s) ligated to the end of the sample nucleic acid molecule, or as part of a primer that binds to a sequence at the end of the sample nucleic acid fragment, or an adapter such as a universal adapter added thereto, as further described herein, the entire nucleic acid molecule can be a sample nucleic acid fragment. In other embodiments, for example, in certain embodiments where an adapter is added to the sample nucleic acid molecule as part of a primer that targets an internal binding site at the end of the sample nucleic acid molecule, a portion of the sample nucleic acid molecule can be a sample nucleic acid fragment targeted for downstream sequencing. For example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sample nucleic acid molecule can be a nucleic acid fragment.

[0080] In some embodiments, the sample nucleic acid molecules are a mixture of nucleic acids isolated from natural sources, with some sample nucleic acid molecules having identical sequences, some sharing at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity, and some having less than 50%, 40%, 30%, 20%, 10%, or 5% sequence identity across a range of 20, 25, 50, 75, 100, 125, 150, 200, or 250 nucleotides at the lower end to a range of 50, 75, 100, 125, 150, 200, 250, 300, 400, or 500 nucleotides at the upper end. Such sample nucleic acid molecules may be nucleic acid samples isolated from tissues or bodily fluids of mammals, such as humans, without enriching for certain sequences over others. In other embodiments, target sequences, e.g., from genes of interest, can be enriched prior to performing the methods provided herein.

[0081] Distinguishing between dd-cfDNA and fd-cfDNA In some embodiments, dd-cfDNA is distinguished from fd-cfDNA based on parental genotype.For example, in some embodiments, paternal DNA from the biological father of the fetus is genotyped using the method described herein.In some embodiments, paternal genotype is already known.Based on the genotypes of the paternal and maternal transplant recipients, target loci are selected that are homozygous in both maternal and paternal genomes, and therefore homozygous in the fetal genome.Therefore, the heterozygosity detected in cfDNA at target loci is derived from transplantation and can be used to quantify the amount of dd-cfDNA in biological samples.

[0082] Determining the risk of rejection in transplant recipients In some embodiments, the transplant recipient's risk of rejection is determined using logistic regression, random forest, or decision tree machine learning analysis. In some embodiments, the machine learning analysis incorporates as a parameter the amount of dd-cfDNA in the transplant recipient's sample or a function thereof. In some embodiments, the machine learning analysis incorporates as a parameter the number of dd-cfDNA reads or a function thereof. In some embodiments, the machine learning analysis incorporates as a parameter the estimated ratio of dd-cfDNA to total cfDNA. In some embodiments, the machine learning analysis incorporates as a parameter the amount of dd-cfDNA, the number of dd-cfDNA reads, or the estimated ratio of dd-cfDNA to total cfDNA in the transplant recipient's sample.

[0083] Machine learning can be used to determine whether rejection or non-rejection occurs.Machine learning is disclosed in WO2020 / 018522, which is filed on July 16, 2019 as PCT / US2019 / 041981 and is entitled "Methods and Systems for calling Ploidy States using a Neural Network", and is incorporated herein by reference in its entirety.In some embodiments, the cutoff threshold is scaled according to the amount of total cfDNA in biological sample.

[0084] In some embodiments, the cutoff threshold is expressed as the percentage of dd-cfDNA in the sample (dd-cfDNA%). In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA per volume unit of blood sample. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA per volume unit of blood sample multiplied by the transplant recipient's body weight, BMI, or blood volume.

[0085] In some embodiments, the cutoff threshold takes into account the patient's weight, BMI, or blood volume. In some embodiments, the cutoff threshold takes into account one or more of donor genome copies per volume of plasma, fetal genome copies per volume of plasma, number of fetuses present, gestational stage, cfDNA yield per volume of plasma, donor height, donor weight, donor age, donor sex, donor ethnicity, donor organ mass, donor organ, living vs. deceased donor, familial relationship (or lack thereof) between donor and recipient, recipient height, recipient weight, recipient age, recipient sex, recipient ethnicity, creatinine, eGFR (estimated glomerular filtration rate), cfDNA methylation, DSA (donor-specific antibodies), KDPI (Kidney Donor Profile Index), medications (immunosuppressants, steroids, blood thinners, etc.), infections (BKV, EBV, CMV, UTI), recipient, fetal, and / or donor HLA allele or epitope mismatch, Banff classification of kidney transplant pathology, and legitimate vs. surveillance or protocol biopsy.

[0086] In some embodiments, the method is at least 50% specific and has a 95% confidence interval for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample. In some embodiments, the method is at least 60% specific and has a 95% confidence interval for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample. In some embodiments, the method is at least 70% specific and has a 95% confidence interval for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample. In some embodiments, the method is at least 75% specific and has a 95% confidence interval for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample. In some embodiments, the method is at least 80% specific, with a 95% confidence interval, for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample. In some embodiments, the method is at least 85% specific, with a 95% confidence interval, for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample. In some embodiments, the method is at least 90% specific, with a 95% confidence interval, for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample.In some embodiments, the method has at least 95% specificity, with a 95% confidence interval, in identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the biological sample.

[0087] Some embodiments use either a fixed threshold of dd-cfDNA per plasma volume, or a non-fixed threshold, such as one adjusted or scaled as described herein. The method for determining this may be based on building an algorithm that maximizes performance using a training data set. Other data, such as patient weight, age, or other clinical factors described herein, may also be taken into account.

[0088] In some embodiments, the method further comprises using the amount of dd-cfDNA to determine the occurrence or likelihood of transplant rejection. In some embodiments, the amount of dd-cfDNA is compared to a cutoff threshold to determine the occurrence or likelihood of transplant rejection, the cutoff threshold being adjusted or scaled according to the amount of total cfDNA. In some embodiments, the cutoff threshold is a function of the number of dd-cfDNA reads.

[0089] In some embodiments, the method includes applying a scale or dynamic threshold metric that takes into account the amount of total cfDNA in the sample to more accurately assess transplant rejection. In some embodiments, the method further includes flagging the sample if the amount of total cfDNA is above a predetermined value. In some embodiments, the method further includes flagging the sample if the amount of total cfDNA is below a predetermined value.

[0090] In some embodiments, the machine learning analysis further incorporates time since transplant as a parameter. In some embodiments, the machine learning analysis further incorporates age of the transplant recipient and / or transplant donor as a parameter. In some embodiments, the machine learning analysis further incorporates stage of pregnancy as a parameter. In some embodiments, the machine learning analysis further incorporates number of fetuses as a parameter. In some embodiments, the machine learning analysis further incorporates gender of the transplant recipient, gender(s) of the fetus(s), and / or gender of the transplant donor as parameters.

[0091] In some embodiments, the risk of rejection of the transplant recipient is determined with a sensitivity of at least 0.81, or at least 0.82, or at least 0.83, or at least 0.84, or at least 0.85, or at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90. In some embodiments, the risk of rejection of the transplant recipient is determined with a specificity of at least 0.81, or at least 0.82, or at least 0.83, or at least 0.84, or at least 0.85, or at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90. In some embodiments, the risk of rejection for a transplant recipient is determined by an area under the curve (AUC) of at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90, or at least 0.91, or at least 0.92, or at least 0.93, or at least 0.94, or at least 0.95.

[0092] Method for measuring the amount of nucleic acid In some embodiments, the amount of cfDNA is measured by quantitative PCR. In some embodiments, the amount of cfDNA is measured by real-time PCR. In some embodiments, the amount of cfDNA is measured by digital PCR. In some embodiments, the amount of cfDNA is measured by sequencing, such as high-throughput sequencing, next-generation sequencing, or sequencing by synthesis.

[0093] In some embodiments, the amount of dd-cfDNA is determined using ratiometric and / or machine learning artificial intelligence comparisons at single or multiple time points.

[0094] In some embodiments, the amount of cfDNA is measured by massively multiplexed PCR (mmPCR) to obtain amplicons containing the biomarkers and sequencing the amplicons.

[0095] In some embodiments, the amount of cfDNA is measured using a microarray. In some embodiments, the amount of cfDNA is measured using molecular barcodes and microscopic imaging (such as NanoString nCounter®).

[0096] In some embodiments, the amount of dd-cfDNA is determined by extracting cfDNA from a biological sample of a pregnant transplant recipient, wherein the extracted cfDNA comprises dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification of the extracted DNA at 10 to 50,000 target loci in a single reaction volume selected based on homozygosity of the parents of the fetus; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of dd-cfDNA based on the sequencing reads.

[0097] In some embodiments, the method is performed without prior knowledge of the donor genotype. In some embodiments, the method does not involve determining the genotype of the transplant donor(s). In some embodiments, the paternal genotype of the fetus is known. In some embodiments, the method involves determining the genotype of the biological father of the fetus.

[0098] In some embodiments, the amount of nucleic acid is measured by target amplification. In some embodiments, the amount of a specific cfDNA target is measured by target amplification. In some embodiments, the target amplification comprises PCR. In some embodiments, the primers for target amplification comprise 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 pairs of forward and reverse PCR primers. In some embodiments, target amplification is performed using 500 to 20,000, 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 primer pairs in a single reaction. This involves performing amplification at 100-20,000, 500-20,000, 1,000-10,000, 200-500, 500-1,000, 1,000-2,000, 2,000-5,000, 5,000-10,000, 10,000-20,000, or 20,000-50,000 target loci to obtain amplification products.

[0099] In some embodiments, the target amplification comprises nested PCR. In some embodiments, the primers for target amplification comprise a first universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 targets. and a second universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 inner target-specific primers. In some embodiments, target amplification is performed using a first universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 target-specific primers. The method includes using a PCR amplifier to amplify 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 target loci in a single reaction volume to obtain amplification products.In some embodiments, target amplification is performed using a second universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 inner target-specific primers. The method includes using a primer to amplify 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 target loci in a single reaction volume to obtain amplification products. In some embodiments, the methods disclosed herein comprise PCR amplification of at least 10, at least 100, at least 500, at least 1000, at least 2000 biomarkers from 10-1000, 100-10000, 200-50000, or 500-20000 RNA biomarkers using at least 10, at least 100, at least 500, at least 1000, at least 2000 from 10-1000, 100-10000, 200-50000, 500-20000 pairs of forward and reverse PCR primers. In some embodiments, step (b) comprises amplifying at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100 target RNA molecules from 2 to 10, 200 to 100, 50 to 500, or 50 to 2000 target RNA molecules using at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100 target RNA molecules from 2 to 10, 200 to 100, 50 to 500, or 50 to 2000 pairs of forward and reverse PCR primers.

[0100] In some embodiments, the method further comprises incorporating tags into the amplification products prior to performing high-throughput sequencing, wherein the tags comprise adapters compatible with sequencing. In some embodiments, the method further comprises adding tags to the extracted DNA prior to performing target amplification, wherein the tags comprise adapters for amplification. In some embodiments, the tags comprise sample-specific barcodes, and the method further comprises pooling the amplification products from multiple samples prior to high-throughput sequencing and sequencing the pool of amplification products together in a single run during high-throughput sequencing.

[0101] In some embodiments, the amount of nucleic acid is determined, for example, by using a tracer nucleic acid or an internal calibration nucleic acid. The terms "tracer nucleic acid" and "internal calibration nucleic acid" are used interchangeably and refer to the composition of a nucleic acid whose length, sequence, nucleotide composition, amount, or biological origin are known in advance. The tracer can be added to a biological sample from a subject to help estimate the amount of total cfDNA in the sample. It can also be added to a reaction mixture other than the biological sample itself.

[0102] Cut-off threshold for determining transplant rejection In some embodiments, the cutoff threshold is an estimated percentage of dd-cfDNA to total cfDNA or a function thereof. In some embodiments, the cutoff threshold is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% dd-cfDNA. In some embodiments, the cutoff threshold is adjusted according to the type of organ being transplanted. In some embodiments, the cutoff threshold is adjusted according to the number of organs being transplanted. In some embodiments, the cutoff threshold is adjusted according to the amount of fd-cfDNA in the sample.

[0103] In some embodiments, the cutoff threshold is proportional to the absolute value of the dd-cfDNA concentration. In some embodiments, the cutoff threshold is the copy number of dd-cfDNA or a function thereof. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA per volume unit of a blood sample. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA per volume unit of a blood sample multiplied by the body weight, BMI, or blood volume of the transplant recipient.

[0104] In some embodiments, the method further comprises longitudinally measuring the amount of cfDNA and the amount of dd-cfDNA in the same transplant recipient, and measuring longitudinal changes in the amount of cfDNA or a function thereof and longitudinal changes in the amount of dd-cfDNA or a function thereof.

[0105] Analysis of donor-derived cell-free DNA for monitoring transplant rejection "Acute rejection (AR)" is a rejection reaction by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically abnormal. Acute rejection is characterized by the recipient's immune cells infiltrating the transplanted tissue and exerting effector functions to destroy the transplanted tissue. The onset of acute rejection is rapid and typically occurs in humans within a few weeks of transplant surgery. Generally, acute rejection can be inhibited or suppressed by immunosuppressive drugs such as rapamycin, cyclosporin A, and anti-CD40L monoclonal antibodies.

[0106] "Chronic transplant rejection or injury" or "CAI" typically occurs in humans within months to years after engraftment, even when immunosuppression of acute rejection is successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can usually be described by a series of specific disorders characteristic of a particular organ. For example, in lung transplants, such disorders include fibroproliferative destruction of the airways (bronchiolitis obliterans); in cardiac tissue transplants, such as heart transplants or valve replacements, such disorders include fibroatherosclerosis; in kidney transplants, such disorders include obstructive nephropathy, nephrosclerosis, and tubulointerstitial nephropathy; and in liver transplants, such disorders include vanishing bile duct syndrome. Chronic rejection is also characterized by ischemic injury, denervation of the transplanted tissue, and immunosuppressant-related hyperlipidemia and hypertension.

[0107] The term "transplant rejection" includes both acute and chronic transplant rejection. The term transplant rejection refers to transplants where the recipient is of the same species as the donor ("allograft rejection") or a different species from the donor ("xenograft rejection"). The term "transplant injury" refers to all modes of graft dysfunction, regardless of pathological diagnosis. The term "organ injury" refers to biomarkers that track organ functional decline, whether the organ is native or transplanted, and regardless of etiology.

[0108] In one aspect, the invention relates to a method of quantifying the amount of dd-cfDNA in a biological sample of a maternal transplant recipient, the method comprising: extracting DNA from the biological sample of the transplant recipient, wherein the DNA comprises dd-cfDNA, fd-cfDNA, and rd-cfDNA; preparing a composition from the cfDNA enriched for one or more selected target loci, wherein the maternal transplant recipient and the biological father of the fetus are homozygous at the selected target loci to ensure homozygosity of the fetus at the selected target loci such that heterozygosity observed in the extracted cfDNA at the selected target loci is derived from the transplant; quantifying the amount of cfDNA extracted and the amount of dd-cfDNA based on heterozygosity at the selected target loci; and determining whether the amount of dd-cfDNA, or a function thereof, exceeds a cutoff threshold indicative of transplant rejection.

[0109] In one aspect, the invention further comprises measuring the amount of dd-cfDNA in a biological sample obtained from the maternal transplant recipient, extracting DNA from the sample obtained from the transplant recipient, wherein the extracted DNA comprises dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification of the extracted DNA at 10 to 50,000 target loci in a single reaction volume; sequencing the amplified DNA to obtain sequencing reads; quantifying the amount of dd-cfDNA based on the sequencing reads; and determining transplant rejection based on whether the amount of dd-cfDNA or a function thereof exceeds a cutoff threshold for cfDNA amount indicative of transplant rejection, wherein transplant rejection is determined based on whether the amount of dd-cfDNA or a function thereof exceeds the cutoff threshold for indicative of transplant rejection.

[0110] In another aspect, the invention relates to a method for quantifying the amount of dd-cfDNA in a biological sample of a maternal transplant recipient, the method comprising: extracting DNA from the biological sample of the transplant recipient, wherein the DNA comprises dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification at 10 to 50,000 or 500 to 50,000 target loci in a single reaction volume using 10 to 50,000 or 500 to 50,000 primer pairs, wherein the target loci include polymorphic loci and non-polymorphic loci, and wherein each primer pair is designed to amplify a target sequence of 100 bp or less; and quantifying the amount of dd-cfDNA in the amplification products.

[0111] In another aspect, the invention relates to a method for detecting dd-cfDNA in a biological sample of a maternal transplant recipient, the method comprising: extracting DNA from the biological sample of the transplant recipient, wherein the DNA comprises dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification at 10 to 50,000 target loci in a single reaction volume using 10 to 50,000 primer pairs, wherein the target loci comprise polymorphic and non-polymorphic loci; sequencing the amplification products by high-throughput sequencing; and quantifying the amount of dd-cfDNA.

[0112] In a further aspect, the invention relates to a method of determining likelihood of transplant rejection in a maternal transplant recipient, the method comprising: extracting cfDNA from a biological sample of the transplant recipient, wherein the cfDNA comprises dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing universal amplification of the extracted cfDNA; performing targeted amplification at 10 to 50,000 target loci in a single reaction volume using 10 to 50,000 primer pairs, wherein the target loci comprise polymorphic and non-polymorphic loci; sequencing the amplification products by high-throughput sequencing; and quantifying the amount of dd-cfDNA in the biological sample, wherein a higher amount of dd-cfDNA indicates a higher likelihood of transplant rejection.

[0113] In some embodiments, the method further includes performing universal amplification of the extracted DNA, in some embodiments, the universal amplification preferentially amplifies dd-cfDNA over rd-cfDNA and fd-cfDNA discarded from the ruptured white blood cells.

[0114] In some embodiments, the transplant recipient is a mammal. In some embodiments, the transplant recipient is a human. In some embodiments, the transplant donor is a human, a pig, a primate, a baboon, a cow, or a dog.

[0115] In some embodiments, the transplant recipient is receiving a transplant selected from an organ transplant, a tissue transplant, a cell transplant, and a bodily fluid transplant. In some embodiments, the transplant recipient is receiving a transplant selected from a kidney transplant, a liver transplant, a pancreas transplant, an intestinal transplant, a heart transplant, a lung transplant, a heart / lung transplant, a stomach transplant, a testis transplant, a penis transplant, an ovary transplant, a uterus transplant, a thymus transplant, a face transplant, a hand transplant, a leg transplant, a bone transplant, a bone marrow transplant, a cornea transplant, a skin transplant, a pancreatic islet cell transplant, a heart valve transplant, a blood vessel transplant, and a blood transfusion. In some embodiments, the transplant recipient is receiving a SPK transplant.

[0116] In some embodiments, the quantifying step comprises determining the proportion of dd-cfDNA among the sum of dd-cfDNA, fd-cfDNA, and rd-cfDNA in the biological sample. In some embodiments, the quantifying step comprises determining the number of copies of dd-cfDNA per unit volume of the blood sample.

[0117] In some embodiments, the method further comprises using the quantified amount of dd-cfDNA to detect the occurrence or likelihood of occurrence of active rejection of the transplant. In some embodiments, the method is performed without prior knowledge of the donor genotype.

[0118] In some embodiments, target amplification involves simultaneously amplifying 10-50,000 target loci in a single reaction volume using (i) at least 10-50,000 different primer pairs, or (ii) at least 10-50,000 target-specific primers and 10-50,000 primer pairs of universal or tag-specific primers.

[0119] In some embodiments, each primer pair is designed to amplify a target sequence of approximately 50-100 bp. In some embodiments, each primer pair is designed to amplify a target sequence of 75 bp or less. In some embodiments, each primer pair is designed to amplify a target sequence of approximately 60-75 bp. In some embodiments, each primer pair is designed to amplify a target sequence of approximately 65 bp.

[0120] In some embodiments, target amplification comprises amplifying at least 10 target loci in a single reaction volume. In some embodiments, target amplification comprises amplifying at least 100 target loci in a single reaction volume. In some embodiments, target amplification comprises amplifying at least 1,000 target loci in a single reaction volume. In some embodiments, target amplification comprises amplifying at least 2,000 target loci in a single reaction volume. In some embodiments, target amplification comprises amplifying at least 5,000 target loci in a single reaction volume. In some embodiments, target amplification comprises amplifying at least 10,000 target loci in a single reaction volume. In some embodiments, target amplification comprises amplifying 10 to 10,000, 10 to 50,000, 100 to 50,000, or 1000 to 50,000 target loci in a single reaction volume.

[0121] In some embodiments, the method further comprises measuring the abundance of one or more alleles at a target locus that is a polymorphic locus. In some embodiments, the polymorphic locus and the non-polymorphic locus are amplified in a single reaction.

[0122] In some embodiments, the quantifying step comprises detecting the amplified target loci using a microarray. In some embodiments, the quantifying step does not comprise using a microarray.

[0123] In some embodiments, target amplification involves simultaneously amplifying 10-50,000 target loci in a single reaction volume using (i) at least 10-50,000 different primer pairs, or (ii) at least 10-50,000 target-specific primers and 10-50,000 primer pairs of universal or tag-specific primers.

[0124] In a further aspect, the present invention relates to a method for diagnosing transplant as acute rejection in a maternal transplant recipient, the method comprising extracting DNA from a biological sample of the maternal transplant recipient, the DNA comprising dd-cfDNA and rd-cfDNA; performing universal amplification of the extracted DNA; and performing targeted amplification at 100 to 50,000 target loci in a single reaction volume using 100 to 50,000 primer pairs, the target loci being polymorphic genetic loci. and quantifying the amount of dd-cfDNA in the biological sample, wherein an amount of dd-cfDNA greater than 1% (or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1.1%, or 1.2%, or 1.3%, or 1.4%, or 1.5%, or 1.6%, or 1.7%, or 1.8%, or 1.9%, or 2.0%) indicates acute transplant rejection.

[0125] In some embodiments, the transplant rejection is antibody-mediated transplant rejection. In some embodiments, the transplant rejection is T-cell-mediated transplant rejection.

[0126] In some embodiments, an amount of dd-cfDNA less than 1% (or 0.9%, or 0.8%, or 0.7%, or 0.6%, or 0.5%) indicates that the transplant will undergo borderline rejection, other damage, or will be stable.

[0127] In a further aspect, the invention relates to a method of monitoring immunosuppressive therapy in a subject, the method comprising: extracting DNA from a biological sample of a transplant recipient, wherein the DNA comprises dd-cfDNA, rd-cfDNA, and fd-cfDNA; performing universal amplification of the extracted DNA; performing targeted amplification at 10 to 50,000 target loci in a single reaction volume using 10 to 50,000 primer pairs, wherein the target loci comprise polymorphic and non-polymorphic loci; sequencing the amplification products by high-throughput sequencing; and quantifying the amount of dd-cfDNA in the biological sample, wherein a change in the level of dd-cfDNA over a time interval is indicative of transplant status.

[0128] In some embodiments, the method further comprises adjusting immunosuppressive therapy based on the level of dd-cfDNA over the time interval.

[0129] In some embodiments, an increased level of dd-cfDNA indicates transplant rejection and the need for adjustment of immunosuppressive therapy. In some embodiments, an unchanged or decreased level of dd-cfDNA indicates transplant tolerance or stability and the need for adjustment of immunosuppressive therapy.

[0130] In some embodiments, the method does not include determining the genotype of the transplant donor and / or transplant recipient.

[0131] In some embodiments, the target loci include at least 10 polymorphic loci, or at least 100 polymorphic loci, or at least 1,000 polymorphic loci, or at least 2,000 polymorphic loci, or at least 5,000 polymorphic loci, or at least 10,000 polymorphic loci.

[0132] In some embodiments, the extraction step includes size selection to enrich for dd-cfDNA and reduce the amount of rd-cfDNA discarded from the ruptured leukocytes.

[0133] In some embodiments, the universal amplification step preferentially amplifies dd-cfDNA over rd-cfDNA or fd-cfDNA.

[0134] In some embodiments, the method includes longitudinally collecting multiple blood samples from the transplant recipient after transplantation and measuring the amount of cfDNA and dd-cfDNA to determine longitudinal changes in the amount of cfDNA or a function thereof, and longitudinal changes in the amount of dd-cfDNA in the transplant recipient. In some embodiments, the method includes collecting and analyzing blood samples from the transplant recipient over a period of about 3 months, about 6 months, about 12 months, about 18 months, or about 24 months, etc. In some embodiments, the method includes collecting blood samples from the transplant recipient at intervals of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, or about 3 months, etc.

[0135] In some embodiments, the method further comprises titrating the dosage of the immunosuppressive therapy according to the longitudinal change in the total amount of cfDNA or a function thereof and the longitudinal change in the amount of dd-cfDNA or a function thereof.

[0136] In one aspect, the disclosure relates to a method of administering immunosuppressive therapy in a maternal transplant recipient, the method comprising: measuring the amount of cfDNA in a biological sample from the transplant recipient; measuring the amount of dd-cfDNA in the biological sample from the transplant recipient; and titrating the dosage of the immunosuppressive therapy according to the amount of cfDNA or a function thereof, and the amount of dd-cfDNA or a function thereof.

[0137] In some embodiments, the method has a sensitivity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in distinguishing acute rejection (AR) from non-AR at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0138] In some embodiments, the method has a specificity of at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% in distinguishing AR from non-AR at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0139] In some embodiments, the method has an area under the curve (AUC) of at least 0.8, or 0.85, or at least 0.9, or at least 0.95 in distinguishing AR from non-AR at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0140] In some embodiments, the method has a sensitivity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in distinguishing AR from normal stable allografts (STA) at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0141] In some embodiments, the method has a specificity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in distinguishing AR over STA at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0142] In some embodiments, the method has an AUC of at least 0.8, or 0.85, or at least 0.9, or at least 0.95, or at least 0.98, or at least 0.99 in distinguishing AR over STA at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0143] In some embodiments, the method has a sensitivity determined by a limit of blank (LoB) of 0.5% or less and a limit of detection (LoD) of 0.5% or less. In some embodiments, the LoB is 0.23% or less and the LoD is 0.29% or less. In some embodiments, the sensitivity is further determined by the limit of quantitation (LoQ). In some embodiments, the LoQ may be 10-fold greater than the LoD, the LoQ may be 5-fold greater than the LoD, the LoQ may be 1.5-fold greater than the LoD, the LoQ may be 1.2-fold greater than the LoD, the LoQ may be 1.1-fold greater than the LoD, or the LoQ may be equal to or greater than the LoD. In some embodiments, the LoB is 0.04% or less, the LoD is 0.05% or less, and / or the LoQ is equal to the LoD.

[0144] In some embodiments, the method has an accuracy determined by evaluating a linear value obtained from a linear regression analysis of the measured donor fraction as a function of the corresponding attempted spike level, the linear value being R 2 is the value of R 2 In some embodiments, the value of R 2 The value is 0.999. In some embodiments, the method has an accuracy determined by calculating a slope value and an intercept value using linear regression for the measured donor fraction as a function of the corresponding attempted spike level, where the slope value is from about 0.9 to about 1.2 and the intercept value is from about -0.0001 to about 0.01. In some embodiments, the slope value is about 1 and the intercept value is about 0.

[0145] In some embodiments, the method has a precision determined by calculating the coefficient of variation (CV), wherein the CV is less than about 10.0%. The CV is less than about 6%. In some embodiments, the CV is less than about 4%. In some embodiments, the CV is less than about 2%. In some embodiments, the CV is less than about 1%.

[0146] In some embodiments, the AR is antibody-mediated rejection (ABMR). In some embodiments, the AR is T-cell-mediated rejection (TCMR).

[0147] In some embodiments, disclosed herein are methods for amplifying target loci in dd-cfDNA from a biological sample of a maternal transplant recipient, the method comprising: extracting DNA from a blood sample of the transplant recipient, the DNA including DNA from transplanted cells, the transplant recipient, and the fetus; enriching the extracted DNA at target loci, the target loci including 10 to 50,000 target loci, including polymorphic and non-polymorphic loci; and amplifying the target loci.

[0148] In some embodiments, disclosed herein are methods for detecting dd-cfDNA in a blood sample from a maternal transplant recipient, the method comprising: (a) extracting DNA from the transplant recipient's blood sample, the DNA including cfDNA derived from both the transplanted cells and the transplant recipient; (b) enriching the extracted DNA at target loci, the target loci including 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; (c) amplifying the target loci; (d) contacting the amplified target loci with probes that specifically hybridize to the target loci; and (e) detecting binding of the probes to the target loci, thereby detecting dd-cfDNA in the blood sample. In some embodiments, the probes are labeled with a detectable marker.

[0149] In some embodiments, disclosed herein are methods for detecting potential transplant rejection in a maternal transplant recipient, the method comprising: a) extracting DNA from a blood sample of the transplant recipient, the DNA including cfDNA from both the transplanted cells and the transplant recipient; b) enriching the extracted DNA at target loci, the target loci including 50-5000 target loci, including polymorphic and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplant DNA and the amount of transplant recipient DNA in the recipient blood sample, wherein a higher amount of dd-cfDNA indicates a higher potential for transplant rejection.

[0150] In some embodiments, disclosed herein are methods for monitoring immunosuppressive therapy in a subject, the methods comprising: a) extracting DNA from a biological sample of a transplant recipient, the DNA including cfDNA derived from transplanted cells, the transplant recipient, and the fetus; b) enriching the extracted DNA at target loci, the target loci including 10,000 to 50,000 target loci, the target loci including polymorphic and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplant DNA and the amount of recipient and fetal DNA in the recipient biological sample, wherein a change in the level of dd-cfDNA over a time interval indicates transplant status. In some embodiments, the method further comprises adjusting the immunosuppressive therapy based on the level of dd-cfDNA over a time interval. In some embodiments, an increase in the level of dd-cfDNA indicates transplant rejection and the need for adjustment of immunosuppressive therapy. In some embodiments, a change or decrease in the level of dd-cfDNA indicates transplant tolerance or stability and the need for adjustment of immunosuppressive therapy.

[0151] In some embodiments, the method disclosed herein requires that the target locus is homozygous in the maternal transplant recipient and the biological father of the fetus, and ensures the homozygosity of the target locus in the fetus.The heterozygosity detected in the biological sample from the maternal transplant recipient is derived from the donor.Therefore, the heterozygosity at the target locus can be used to quantify the amount of dd-cfDNA present in the biological sample.

[0152] Analysis method In some embodiments, the method also includes obtaining genotype data from one or more of the transplant donor, maternal transplant recipient, fetus, and the biological father of the fetus. In some embodiments, obtaining genotype data from one or more of the transplant donor, maternal transplant recipient, fetus, and the biological father of the fetus includes preparing DNA from the donor, recipient, fetus, and father, where the preparation includes preferentially enriching DNA at a plurality of polymorphic loci to obtain prepared DNA, optionally amplifying the prepared DNA, and measuring DNA in the prepared sample at the plurality of polymorphic loci. The genotype data can be used to determine loci at which the maternal transplant recipient and the biological father of the fetus are homozygous.

[0153] In some embodiments, constructing a joint distribution model of expected allele count probabilities for multiple polymorphic loci on a chromosome is performed using genotype data obtained from one or more of the transplant donor, the maternal transplant recipient, the fetus, and the biological father of the fetus. In some embodiments, the first sample is isolated from the plasma of the transplant recipient, and obtaining the genotype data from the transplant recipient is performed by estimating the recipient's genotype data from DNA measurements made on the prepared sample.

[0154] In some embodiments, the preferential enrichment results in an average degree of allelic bias between the prepared sample and the first sample of a factor selected from the group consisting of 2-fold or less, 1.5-fold or less, 1.2-fold or less, 1.1-fold or less, 1.05-fold or less, 1.02-fold or less, 1.01-fold or less, 1.005-fold or less, 1.002-fold or less, 1.001-fold or less, and 1.0001-fold or less. In some embodiments, the plurality of polymorphic loci are SNPs. In some embodiments, measuring the DNA in the prepared sample is performed by sequencing.

[0155] In some embodiments, a diagnostic box is disclosed to aid in determining the transplant status of a maternal transplant recipient, where the diagnostic box can perform the preparation and measurement steps of the disclosed methods.

[0156] In some embodiments, the allele counts are probabilistic rather than binary. In some embodiments, measurements of DNA from samples prepared at multiple polymorphic loci are also used to determine whether a transplant has inherited one or more linked haplotypes.

[0157] In some embodiments, constructing a joint distribution model of allele count probabilities is done by modeling the dependency between polymorphic alleles on a chromosome using data on the probability of chromosome crossover at different locations on the chromosome. In some embodiments, constructing a joint distribution model of allele counts and determining the relative probability of each hypothesis is done using a method that does not require the use of a reference chromosome.

[0158] In some embodiments, determining the relative probability of each hypothesis utilizes the estimated proportion of dd-cfDNA in the prepared sample. In some embodiments, the DNA measurements from the prepared sample used to calculate allele count probabilities and determine the relative probability of each hypothesis include primary genotype data. In some embodiments, selecting the transplant state corresponding to the hypothesis with the greatest probability is performed using maximum likelihood estimation or maximum a posteriori estimation.

[0159] In some embodiments, calling the transplantation status also includes combining the relative probability of each status hypothesis determined using a joint distribution model and the allele count probabilities with the relative probability of each status hypothesis calculated using statistical methods derived from the group consisting of read count analysis, heterozygosity rate comparisons, statistics available only using donor genetic information, normalized genotype signal probabilities for a particular donor / recipient context, statistics calculated using estimated transplantation fractions for the first sample or prepared sample, and combinations thereof.

[0160] In some embodiments, a reliability estimate is calculated for the called transplant status. In some embodiments, the method also includes taking a clinical action based on the called transplant status.

[0161] In some embodiments, a report indicating the determined transplantation status is generated using this method. In some embodiments, a kit for determining transplantation status designed for use with the methods disclosed herein is disclosed, the kit including a plurality of inner forward primers and optionally a plurality of inner reverse primers, each primer designed to hybridize to a DNA region immediately upstream and / or downstream from one of the polymorphic sites on the target chromosome, and optionally to additional chromosomes, wherein the hybridization region is separated from the polymorphic site by a small number of bases, the small number being selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-60, and combinations thereof.

[0162] In some embodiments, the cutoff threshold takes into account one or more of donor genome copies per volume of plasma, cfDNA yield per volume of plasma, fetal genome copies per volume of plasma, number of fetuses present, gestational stage, donor height, donor weight, donor age, donor sex, donor ethnicity, donor organ weight, donor organ, living vs. deceased donor, related vs. unrelated donor, recipient height, recipient weight, recipient age, recipient sex, recipient ethnicity, creatinine, eGFR (estimated glomerular filtration rate), cfDNA methylation, DSA (donor-specific antibodies), KDPI (Kidney Donor Profile Index), medications (immunosuppressants, steroids, anticoagulants, etc.), infections (BKV, EBV, CMV, UTI), recipient and / or donor HLA allele or epitope mismatch, Banff classification of kidney transplant pathology, and cause-specific vs. surveillance or protocol biopsy.

[0163] In some embodiments, the cutoff threshold is scaled according to the amount of total cfDNA in the blood sample.

[0164] In some embodiments, the method has a sensitivity of at least 80% with a confidence interval of 95% in identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0165] In some embodiments, the method has a sensitivity of at least 70% with a confidence interval of 95% in identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0166] In some embodiments, the method has a sensitivity of at least 80% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 85% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 90% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 95% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0167] In some embodiments, the method has a sensitivity of at least 70% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 75% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 85% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 90% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 95% with a confidence interval of 95% in identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0168] Multiplex Amplification In some embodiments, the method includes performing a multiplex amplification reaction to amplify multiple target loci in a single reaction mixture prior to sequencing the selectively enriched DNA. In some embodiments, the target loci are selected at loci for which both the maternal transplant recipient and the biological father of the fetus are homozygous.

[0169] In certain exemplary embodiments, nucleic acid sequence data is generated by performing high-throughput sequencing of multiple copies of a series of amplicons generated using a multiplex amplification reaction, where each amplicon in the series spans at least one polymorphic locus in a set of polymorphic loci, and each of the polymorphic loci in the set is amplified. For example, in these embodiments, multiplex PCR may be performed to amplify amplicons across at least 10, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, or 100,000 polymorphic loci (e.g., SNP loci). This multiplex reaction can be configured as a single reaction or as a pool of multiplex reactions of different subsets. The multiplex reaction methods provided herein, such as the massively multiplexed PCR disclosed herein, provide exemplary processes for performing amplification reactions to help achieve improved multiplexing and, therefore, sensitivity levels.

[0170] In some embodiments, the amplification is performed using direct multiplex PCR, sequential PCR, nested PCR, double nested PCR, one-and-a-half PCR, or any combination thereof. sided nested PCR, fully nested PCR, one-sided fully nested PCR, one-sided nested PCR, hemi-nested PCR, hemi-nested PCR, triplex hemi-nested PCR, semi-nested PCR, one-sided semi-nested PCR, reverse semi-nested PCR, or one-sided PCR, as described in U.S. Application No. 13 / 683,604, filed November 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed November 18, 2011, U.S. Publication No. 2012 / 0270212, and U.S. Serial No. 61 / 994,791, filed May 16, 2014, which are incorporated by reference in their entireties.

[0171] In some embodiments, multiplex PCR is used. In some embodiments, a method for amplifying target loci in a nucleic acid sample includes (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 10, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, or 100,000 different target loci to generate a single reaction mixture, and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to generate amplified products containing target amplicons. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplified products are primer dimers. In some embodiments, the primers are in solution (e.g., dissolved in a liquid phase rather than a solid phase). In some embodiments, the primers are in solution and not immobilized on a solid support. In some embodiments, the primers are not part of a microarray.

[0172] In certain embodiments, the multiplex amplification reaction is performed under limiting primer conditions for at least half of the reactions. In some embodiments, limiting primer concentrations are used in 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or all of the reactions in the multiplex reaction. Factors to consider in achieving limiting primer conditions in amplification reactions such as PCR are provided herein.

[0173] For certain embodiments, the multiplex amplification reaction may include, for example, 2,500 to 50,000 multiplex reactions. In certain embodiments, multiplex reactions ranging from 10, 100, 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, and 50,000 at the lower end of the range to 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, 50,000, and 100,000 at the higher end of the range are performed.

[0174] In one embodiment, multiplex PCR assays are designed to amplify potentially heterozygous SNP loci or other polymorphic or non-polymorphic loci on one or more chromosomes, and these assays are used in a single reaction to amplify DNA. The number of PCR assays can be 10 to 200 PCR assays, 200 to 1,000 PCR assays, 1,000 to 5,000 PCR assays, or 5,000 to 20,000 PCR assays (10 to 200 reactions, 200 to 1,000 reactions, 1,000 to 5,000 reactions, 5,000 to 20,000 reactions, and more than 20,000 reactions, respectively). In one embodiment, a multiplex pool of at least about 10,000 PCR assays (10,000 reactions) is designed to amplify potentially heterozygous SNP loci in a single reaction and amplify cfDNA obtained from blood, plasma, serum, solid tissue, or urine samples. The SNP frequency of each locus can be determined by clonal methods or some other method of sequencing the amplicon. In another embodiment, the original cfDNA sample is split into two samples and 5,000 parallel assays are performed. In another embodiment, the original cfDNA sample is split into n samples and approximately 10,000 / n parallel assays are performed, where n is 2 to 12, or 12 to 24, or 24 to 48, or 48 to 96.

[0175] In one embodiment, the method disclosed herein uses highly efficient, highly multiplexed targeted PCR to amplify DNA, followed by high-throughput sequencing to determine the allele frequency at each target locus. One technique that allows highly multiplexed targeted PCR to be performed efficiently involves designing primers that are unlikely to hybridize with each other. PCR probes, typically called primers, are selected by creating a thermodynamic model of potentially harmful interactions between at least 10, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 potential primer pairs or unintended interactions between primers and sample DNA, and then using this model to eliminate designs that are incompatible with other designs in the pool. Another technique that allows highly multiplexed targeted PCR to be performed efficiently is to use a partial or full nesting approach to targeted PCR. Using one or a combination of these approaches allows for the multiplexing of at least 10, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 primers in a single pool, with the resulting amplified DNA containing the majority of DNA molecules, when sequenced, mapping to the target locus. Using one or a combination of these techniques allows for the multiplexing of large numbers of primers in a single pool, with the resulting amplified DNA containing more than 50%, more than 80%, more than 90%, more than 95%, more than 98%, or more than 99% of DNA molecules mapping to the target locus.

[0176] Bioinformatics methods are used to analyze the genotype data obtained from the multiplex PCR. Bioinformatics methods useful and relevant to the methods disclosed herein can be found in U.S. Patent Publication No. 2018 / 0025109, which is incorporated herein by reference.

[0177] High-throughput sequencing In some embodiments, the sequence of the amplicon is determined by performing high-throughput sequencing.

[0178] Genotype data of transplant recipients and / or transplant donors can be converted from molecular to electronic state by measuring the appropriate genetic material using tools and techniques from a group including, but not limited to, genotyping microarrays and high-throughput sequencing. Some high-throughput sequencing methods include Sanger DNA sequencing, pyrosequencing libraries, the ILLUMINA SOLEXA platform, ILLUMINA's genome analyzer, or APPLIED BIOSYSTEM's 454 sequencing platform, HELICOS's TRUE SINGLE MOLECULE SEQUENCING platform, HALCYON MOLECULAR's electron microscope sequencing, PacBio, Oxford Nanopore, or any other sequencing method. In some embodiments, high-throughput sequencing is performed on an Illumina NextSeq®. All of these methods physically convert the genotype data stored in a sample of DNA into a set of genotype data that is typically stored in a memory device during processing.

[0179] In some embodiments, the sequence of the selectively enriched DNA is determined by performing a microarray analysis. In one embodiment, the microarray may be an ILLUMINA SNP microarray or an AFFYMETRIX SNP microarray.

[0180] In some embodiments, the sequence of the selectively enriched DNA is determined by performing quantitative PCR (qPCR) or digital droplet PCR (ddPCR) analysis. qPCR measures the intensity of fluorescence at a specific time (generally per amplification cycle) to determine the relative amount of target molecules (DNA). ddPCR measures the actual number of molecules (target DNA) because each molecule is in a droplet, thus making it a separate "digital" measurement. This provides absolute quantification because ddPCR measures the sample's positivity rate, i.e., the number of droplets that fluoresce due to proper amplification. This positivity rate accurately indicates the initial amount of template nucleic acid.

[0181] definition As used herein, the term "single nucleotide polymorphism (SNP)" refers to a single nucleotide that may differ between the genomes of two members of the same species. The use of this term does not imply any restriction on the frequency with which each variant occurs.

[0182] In some embodiments, for example, sequence refers to a DNA or RNA sequence or a gene sequence. It may refer to the primary physical structure of a DNA or RNA molecule or strand in an individual. It may refer to the sequence of nucleotides present in that DNA or RNA molecule, or the complementary strand of a DNA or RNA molecule. It may refer to the information contained in a DNA or RNA molecule as its in silico representation.

[0183] In some embodiments, for example, a locus refers to a particular region of interest on an individual's DNA or RNA, including, but not limited to, one or more SNPs, potential insertion or deletion sites, or sites of some other associated genetic variation. A disease-associated SNP can also refer to a disease-associated locus.

[0184] In some embodiments, for example, a polymorphic allele, or "polymorphic locus," refers to an allele or locus whose genotype varies between individuals within a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms (SNPs), short tandem repeats, deletions, duplications, and inversions.

[0185] In some embodiments, for example, an allele refers to a nucleotide or nucleotide sequence that occupies a particular locus.

[0186] In some embodiments, for example, genotype data, or "genotype data," refers to data describing aspects of one or more individual genomes. It can refer to one or a set of loci, a partial or entire sequence, a partial or entire chromosome, or the entire genome. It can refer to the identity of one or more nucleotides. It can refer to a set of contiguous nucleotides, or nucleotides at different locations in the genome, or a combination thereof. Genotype data is in silico, although the physical nucleotides in a sequence can also be considered chemically encoded genotype data. Genotype data can be referred to as "pertaining to," "of," "at," "from," or "relating to" an individual(s). Genotype data can refer to output measurements from a genotyping platform where these measurements are made on genetic material.

[0187] In some embodiments, for example, genetic material, or "genetic sample," refers to physical matter, such as tissue or blood, from one or more individuals that contains nucleic acid (including, for example, DNA or RNA).

[0188] In some embodiments, for example, allele type data refers to a set of genotype data for a set of one or more alleles. It can refer to graded haplotype data. It can refer to SNP identities, and it can refer to nucleic acid sequence data, including insertions, deletions, repeats, and mutations.

[0189] In some embodiments, for example, an allele state refers to the actual state of a gene within a set of one or more alleles. This may refer to the actual state of a gene as described by allele type data.

[0190] In some embodiments, for example, allele ratio or allele ratio refers to the ratio between the amount of each allele at a locus present in a sample or individual. If the sample is measured by sequencing, the allele ratio may refer to the proportion of sequence reads that map to each allele at the locus. If the sample is measured by an intensity-based measurement method, the allele ratio may refer to the proportion of the amount of each allele present at that locus as estimated by the measurement method.

[0191] In some embodiments, for example, the allele count refers to the number of sequences that map to a particular locus, or, if the locus is polymorphic, the number of sequences that map to each of the alleles. If each allele is counted in a binary manner, the allele count will be an integer. If alleles are counted probabilistically, the allele count may be a fraction.

[0192] In some embodiments, for example, a primer, or a "PCR probe," refers to a single DNA molecule (DNA oligomer) or a collection of DNA molecules (DNA oligomers) where the DNA molecules are identical or nearly identical, the primer includes a region designed to hybridize to a target polymorphic locus, and includes a priming sequence designed to enable amplification, such as PCR amplification. A primer may also include a molecular barcode. A primer may include a random region that is different for each individual molecule.

[0193] In some embodiments, for example, a hybrid capture probe refers to any nucleic acid sequence, possibly modified, generated by various methods, such as PCR or direct synthesis, that is intended to be complementary to one strand of a specific target DNA or RNA sequence in a sample. An exogenous hybrid capture probe may be added to a prepared sample and hybridized through a denaturation-annealing process to form exogenous-endogenous fragment duplexes. These duplexes can then be physically separated from the sample by various means.

[0194] In some embodiments, for example, a sequence read refers to data representing a sequence of nucleotide bases measured using clonal sequencing methods. Clonal sequencing can generate sequence data representing a single, clone, or cluster of original DNA or RNA molecules. A sequence read can also have an associated quality score for each base position in the sequence, indicating the likelihood that the nucleotide was called correctly.

[0195] In some embodiments, for example, mapping a sequence read is the process of determining the location of the origin of a sequence read in the genome sequence of a particular organism. The location of the origin of a sequence read is based on the similarity of the nucleotide sequences of the read and the genome sequence.

[0196] In some embodiments, for example, DNA or RNA of donor origin refers to DNA or RNA that was originally part of cells whose genotype was essentially equivalent to that of the transplant donor. The donor can be a human or a non-human mammal (e.g., a pig).

[0197] In some embodiments, for example, DNA or RNA of recipient origin refers to DNA or RNA that was originally part of a cell whose genotype was essentially equivalent to that of the transplant recipient.

[0198] In some embodiments, DNA may refer to genomic DNA, cDNA, cell-free DNA (cfDNA), cell-free mitochondrial DNA (cf mDNA), cell-free DNA derived from nuclear DNA (cf nDNA), cellular DNA, or mitochondrial DNA. In some embodiments, cfDNA is derived from exosomes or microvesicles.

[0199] In some embodiments, RNA may refer to messenger RNA (mRNA), small non-coding RNA (sncRNA), transfer RNA (tRNA), or non-protein-coding RNA from a cell. In some embodiments, sncRNA includes microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), or other RNA (miscRNA). In some embodiments, the RNA is cell-free RNA. In some embodiments, the cell-free RNA is derived from exosomes or microvesicles.

[0200] In some embodiments, amplification of RNA involves reverse transcribing the RNA to produce complementary DNA (cDNA), followed by amplification of the cDNA by amplification methods disclosed elsewhere herein.

[0201] In some embodiments, for example, transplant recipient plasma refers to the plasma portion from the blood of a female patient who has received an allograft or xenograft, eg, an organ transplant recipient.

[0202] As used herein, the terms "maternal transplant recipient," "gestational transplant recipient," and "transplant recipient" are used interchangeably. In some embodiments, a maternal transplant recipient refers to a pregnant patient who has received an allograft or xenograft, such as an organ transplant recipient. In some embodiments, a maternal transplant recipient may not be pregnant, but nucleic acids from the fetus, such as fd-cfDNA, may still be present in the maternal transplant recipient. In some embodiments, the biological father of the fetus is the father of the maternal transplant recipient's fetus. In some embodiments, paternal DNA refers to DNA from the biological father of the fetus.

[0203] In some embodiments, the biological sample includes blood, plasma, saliva, semen, sperm, cell culture supernatant, mucus secretion, dental plaque, gastrointestinal tissue, stool, urine, hair, bone, bodily fluids, tears, tissue, skin, nails, germ cells, embryos, amniotic fluid, chorionic villus samples, bile, lymph, cervical mucus, or a forensic sample.

[0204] In some embodiments, for example, preferential enrichment of DNA or RNA corresponding to a locus, or preferential enrichment of DNA or RNA at a locus, refers to any technique that results in a higher proportion of DNA or RNA molecules in a post-enrichment DNA or RNA mixture corresponding to a locus than the proportion of DNA or RNA molecules in the pre-enrichment DNA or RNA mixture corresponding to the locus. The technique may include selective amplification of DNA or RNA molecules corresponding to the locus. The technique may include removing DNA or RNA molecules that do not correspond to the locus. The technique may include a combination of methods. Enrichment is defined as the proportion of DNA or RNA molecules in the post-enrichment mixture that correspond to the locus divided by the proportion of DNA or RNA molecules in the pre-enrichment mixture that correspond to the locus. Preferential enrichment may be performed at multiple loci. In some embodiments of the present disclosure, the enrichment is greater than 20. In some embodiments of the present disclosure, the enrichment is greater than 200. In some embodiments of the present disclosure, the enrichment is greater than 2,000. When preferential enrichment is performed at multiple loci, enrichment may refer to the average enrichment of all loci in the set of loci.

[0205] In some embodiments, for example, amplification refers to techniques that increase the copy number of DNA and / or RNA molecules.

[0206] In some embodiments, for example, selective amplification can refer to techniques that increase the copy number of specific molecules of DNA and / or RNA or molecules of DNA and / or RNA corresponding to specific regions of DNA and / or RNA. It can also refer to techniques that increase the copy number of specific target molecules of DNA and / or RNA or targeted regions of DNA and / or RNA over non-target molecules or regions of DNA and / or RNA. Selective amplification can be a method of preferential enrichment.

[0207] In some embodiments, for example, a universal priming sequence refers to a DNA sequence that can be added to a population of target nucleic acid molecules by, for example, ligation, PCR, or ligation-mediated PCR. Once added to a population of target molecules, primers specific to the universal priming sequence can be used to amplify the target population using a single amplification primer pair. The universal priming sequence does not need to be related to the target sequence.

[0208] In some embodiments, for example, a universal adaptor, or "ligation adaptor" or "library tag," is a DNA molecule that contains universal priming sequences that can be covalently attached to the 5' and 3' ends of a population of target double-stranded DNA molecules. The addition of the adaptor provides universal priming sequences at the 5' and 3' ends of the target population from which PCR amplification can be performed, amplifying all molecules from the target population using a single amplification primer pair.

[0209] In some embodiments, for example, targeting refers to a method used to selectively amplify or otherwise preferentially enrich for DNA or RNA molecules corresponding to a set of loci in a mixture of DNA or RNA. [Example]

[0210] Example 1 This example is illustrative only, and those skilled in the art will appreciate that the invention disclosed herein can be practiced in a variety of other ways.

[0211] blood sample Pregnant female adult or young adult patients receive donor organs from related or unrelated living donors, or from unrelated cadaveric donors. The time of patient blood collection after transplant surgery is either at the time of transplant biopsy or at various pre-specified time intervals based on laboratory protocols. Optionally, samples are matched for biopsy, and blood is collected at the time of clinical dysfunction and biopsy, or at the time of protocol biopsy (at which point most patients have no clinical dysfunction). Additionally, some patients undergo serial blood collection after transplant.

[0212] Measurement of nucleic acids in blood samples The workflow and statistical analysis are disclosed in Sigdel et al., J. Clin. Med. 8(1):19 (2019), which is incorporated herein by reference in its entirety. Nucleic acids, such as RNA or DNA, particularly cfDNA, are extracted from plasma samples using the QIAamp™ Circulating Nucleic Acid Kit (Qiagen), and the LabChip™ NGS 5k Kit (PerkinElmer, Waltham, Massachusetts, USA) is used for quantification. Library preparation is performed using the Natera Library Prep kit, as described in Abbosh et al., Nature 545:446-451 (2017), which is incorporated herein by reference in its entirety, with 18 cycles of library amplification modification to plateau the library. The purified library is quantified using the LabChip™ NGS 5k, as described in Abbosh et al., Nature 545:446-451 (2017). Target enrichment was achieved using massively multiplexed PCR (mmPCR) using a modified version of Zimmermann et al., Prenat. Diagn. 32:1233-1241 (2012), the entire text of which is incorporated herein by reference, targeting 13,392 single nucleotide polymorphisms (SNPs). Amplicons were then single-end sequenced for 50 cycles on an Illumina HiSeq 2500 Rapid Run® at 10-11 million reads per sample.

[0213] Example 2 Determining transplant status in maternal transplant recipients dd-cfDNA was measured in six pregnant kidney recipients, as shown in Figure 1. Identifying SNPs for which both the mother and father were homozygous ensured fetal homozygosity and ensured that any heterozygosity observed in maternal cfDNA was derived from the transplant. These SNPs were used to calculate donor fraction estimates (DFE). Clinical data collected included renal function tests, mode of delivery, preeclampsia, and preexisting hypertension.

[0214] DFE was calculated for four of six kidney transplant recipients. Five of six of these patients had preexisting chronic hypertension, and four of six had severe preeclampsia. All patients underwent medically indicated induction of labor followed by cesarean section delivery for various indications. Chart review also revealed that all patients experienced a spike in serum creatinine and acute kidney injury during the antepartum, postpartum, or intrapartum studies.

[0215] This study demonstrates the feasibility of measuring dd-cfDNA in pregnant kidney recipients with chronic renal dysfunction. These measurements can help distinguish between acute rejection and hypertensive disorders of pregnancy. Larger cohort studies will confirm the utility of longitudinal monitoring during pregnancy to help inform clinicians in clinical decisions that may impact both transplant outcomes and maternal and fetal health.

[0216] Example 3: Donor-derived cell-free DNA (dd-cfDNA) in pregnant kidney transplant recipients Background: Symptoms of early preeclampsia and kidney transplant rejection cannot be distinguished noninvasively in pregnant kidney transplant recipients (KTRs). Donor-derived cell-free DNA (dd-cfDNA) is a biomarker that can be measured noninvasively to assess the risk of allograft rejection. The Prospera™ test measures dd-cfDNA in KTRs using a single-nucleotide polymorphism (SNP)-based massive multiplex PCR (mmPCR) methodology. Detection of dd-cfDNA ≥ 1% is associated with rejection in adult non-pregnant KTRs. Measuring dd-cfDNA in pregnant KTRs requires distinguishing donor cfDNA fragments from fetal and maternal cfDNA fragments. We successfully demonstrated dd-cfDNA measurement in a small cohort of pregnant KTRs with hypertensive disorders, and here we describe two case studies with medical details.

[0217] Methods: Six pregnant KTRs were included in this study. Genomic DNA (gDNA) from the father and total cfDNA from the mother were extracted from cheek swabs and blood samples, respectively (Figure 2A). The proportion of dd-cfDNA in pregnant KTRs was measured using an integrated (Panorama™ / Prospera™) bioinformatics and laboratory workflow incorporating sequencing of maternal total cfDNA and paternal gDNA (Figure 2B). Clinical data were collected, including renal function tests, mode of delivery, serum creatinine, preeclampsia, preexisting hypertension, and medical history.

[0218] Results: dd-cfDNA was calculated for four of six pregnant KTRs. Five of six patients had preexisting chronic hypertension, and four of six had severe preeclampsia. All patients underwent medically indicated induction of labor followed by cesarean section for various indications. All patients experienced spikes in serum creatinine (SCr) and acute kidney injury (AKI) during the prenatal, postnatal, or intrapartum studies. Patient 1 (Figure 3A) was diagnosed with AKI due to tacrolimus toxicity before conception and underwent induction of labor at 38 weeks and 5 days gestation due to elevated blood pressure and SCr levels. Three months before AKI diagnosis, the patient's dd-cfDNA was 1.16%. Patient 2's (Figure 3B) SCr level increased dramatically throughout pregnancy and after delivery. During pregnancy, the patient was suspected of acute KT rejection with hypertension, and her dd-cfDNA was 0.27% around the 15th week of gestation. Due to a possible complication of KT rejection, labor was induced at 27 weeks and 1 day of gestation. After delivery, the patient was diagnosed with AKI accompanied by eclampsia and malignant hypertension due to nonadherence.

[0219] Conclusions: This proof-of-concept study demonstrated the feasibility of measuring dd-cfDNA in pregnant KTRs with chronic renal dysfunction. These measurements can help distinguish between acute rejection and hypertensive disorders of pregnancy. Further validation of dd-cfDNA testing in pregnant KTRs will help inform physicians about allograft and maternal-fetal health.

[0220] Example 4: Donor-derived cell-free DNA (dd-cfDNA) in pregnant kidney transplant recipients Successful kidney transplantation restores fertility, allowing childbearing-age kidney recipients to conceive. Pregnant kidney transplant recipients are at higher risk for maternal-fetal complications, including miscarriage, preterm birth, hypertensive disorders of pregnancy, compromised graft function, and rejection, and kidney transplant rates in this population continue to increase. Graft function is essential for both the pregnant individual and the newborn, and for the life of the allograft. The ability to distinguish between preeclampsia and kidney transplant rejection is crucial because the impact of diagnosis, management, timing of birth, and outcomes vary dramatically during pregnancy.

[0221] Definitive diagnostic testing for kidney rejection requires a renal biopsy, which is only recommended during pregnancy if a histological diagnosis alters management. Traditionally, kidney allograft monitoring included assessment of serum creatinine levels, blood pressure monitoring, and assessment of proteinuria. These parameters serve as indirect indicators of changes in allograft function and may suggest the possibility of acute rejection. However, these parameters are relatively nonspecific markers and may be affected by factors other than rejection, such as dehydration, gestational age, and preeclampsia. Recently, donor-derived cell-free DNA (dd-cfDNA) has emerged as a noninvasive, specific biomarker for allograft rejection.

[0222] Distinguishing between allograft rejection, kidney disease progression, and preeclampsia in pregnant kidney transplant recipients without renal biopsy is currently not feasible using noninvasive methods. Monitoring allograft status in pregnant kidney transplant recipients (KTRs) using dd-cfDNA requires the ability to distinguish between fetal / placental, donor, and recipient cfDNA fragments. This study aims to measure donor-derived cfDNA in a cohort of pregnant kidney transplant recipients using a combination of placenta-derived cfDNA and transplant monitoring techniques using dd-cfDNA in conjunction with paternal DNA swabs. This study demonstrates the feasibility of utilizing dd-cfDNA during pregnancy from a cohort of pregnant individuals with hypertensive disorders.

[0223] Methods: This study included blood samples from pregnant women who received kidney transplants during pregnancy. Prospera™ (Natera, Inc.) was administered to pregnant patients with kidney allografts (n = 9). Prospera™ is a transplant rejection detection and monitoring test that detects allograft donor-derived cell-free DNA (dd-cfDNA) in the blood. dd-cfDNA is expressed as a percentage of total cell-free DNA (cfDNA). This clinical test is a massively multiplex PCR (mmPCR) single-nucleotide polymorphism (SNP)-based genetic test that detects over 13,000 SNPs to accurately measure dd-cfDNA. Prospera for kidney rejection uses a cutoff of ≥1% dd-cfDNA percentage to indicate active rejection. However, calculating donor fraction estimates is more complicated in pregnant women because cfDNA in the mother's peripheral blood is a mixture of maternal cfDNA, donor-derived cfDNA from the transplanted organ, and fetal cfDNA of both maternal and paternal origin. To overcome this issue, we first identified paternal genotypes by sequencing paternal cheek swabs using a modified Prospera™ workflow with genomic DNA (gDNA) as input. Genomic DNA (n = 6) was isolated using the Qiagen DNeasy Blood and Tissue Kit and quantified using a Qubit dsDNA HS (Thermo). gDNA was sheared to approximately 160 bp using a Covaris LE220 ultrasonicator. Sheared samples were quantified, normalized, and libraries prepared. After mmPCR, barcoded samples were pooled, quantified, sized, and sequenced on a 2100 Bioanalyzer (Agilent) using a NextSeq500. Sequencing data was processed through a customized Prospera™ bioinformatics pipeline.

[0224] Determination of donor-derived cfDNA from transplanted kidneys Donor-derived cell-free DNA (dd-cfDNA) was measured in six pregnant kidney recipients by sequencing the patient's cfDNA using the Prospera™ lab workflow, starting with a peripheral blood sample (Figure 2). Custom bioinformatics analysis was performed to quantify donor-derived cfDNA (Figure 4). To determine maternal genotype, any homozygous SNP with more than 75% of reads belonging to one allele is considered homozygous for the mother. To determine paternal genotype, any SNP from the paternal cheek swab with more than 99% of reads belonging to one allele is considered homozygous. If both maternal and paternal genotypes are homozygous for the same allele, the fetal genotype must also be similar. After filtering SNPs for which the mother and fetus have all four copies of the same allele, the Prospera™ algorithm is run on the filtered SNP set to determine the proportion of dd-cfDNA (Figure 4). Clinical data collected included renal function tests, mode of delivery, preeclampsia, and pre-existing hypertension.

[0225] Results: Nine pregnant women were recruited. Paternal cheek swabs were received and available from six of the nine participants, with known and confirmed fathers. These six participants were included in the study. Detailed details are shown in Table 1. Two adjudicated representative cases are shown in Figure 4. Donor-derived cfDNA analysis using Prospera, using a customized data analysis approach, resulted in rejection risk for four patients (Figure 5). In two cases, the donor cfDNA could not be distinguished because the donor was either the fetus's father or the recipient's twin sister. Of the remaining four cases, the DFE was reported as low risk because it was significantly below 1% in three cases, but exceeded the 1% threshold in case 2. Case 2 also had an abnormally high total cfDNA load.

[0226] Discussion: Pregnancy is rare in women undergoing dialysis, with a low incidence of conception (0.9–7%) and a very high rate of preterm birth. Deteriorating renal function can impair fertility in these women, and kidney transplantation has a restorative effect on ovulation and fertility. However, although most pregnancies after kidney transplantation result in live births, there is a significant risk of fetal complications, including preterm birth, low birth weight, and fetal growth restriction (FGR). Evidence regarding live birth rates in pregnant kidney transplant recipients is conflicting, with some studies reporting rates comparable to the general population and others reporting lower rates. Maintaining adequate immunosuppression during pregnancy and adjusting for pregnancy-related pharmacokinetic and physiologic changes is crucial.

[0227] For these reasons, close monitoring by both a transplant nephrologist and a high-risk obstetrician is recommended for pregnant kidney transplant recipients. Monitoring includes surveillance for hypertension, preeclampsia, gestational diabetes, renal allograft dysfunction, and infection, with the primary goal of achieving a near-term or full-term pregnancy without hypertensive complications, graft dysfunction, or rejection.

[0228] The use of cell-free DNA (cfDNA) in prenatal care has gained significant interest in recent years. In the context of pregnancy, cfDNA, also known as noninvasive prenatal testing, allows for the detection of fetal aneuploidies (specifically, trisomies 21, 18, and 13, as well as sex chromosomes). Furthermore, monitoring donor-derived cfDNA (dd-cfDNA) has shown promise as a noninvasive method for detecting early signs of graft rejection in transplant recipients. Studies have shown that dd-cfDNA levels above 1% are associated with an increased likelihood of acute kidney rejection.

[0229] Given these observations, it is reasonable to hypothesize that there is a correlation between the proportion of dd-cfDNA and the occurrence of rejection in pregnant kidney recipients. Serial monitoring of dd-cfDNA levels may identify individuals at high risk for these complications, allow for closer surveillance and timely intervention, and serve as a useful tool for distinguishing acute rejection from preeclampsia without the need for renal biopsy. In this study, we successfully measured dd-cfDNA in a cohort of pregnant kidney transplant recipients using transplant monitoring techniques using dd-cfDNA and paternal buccal DNA swabs. In two cases, the donor was a two-haplotype sister of the child's mother or father. Of the remaining four cases, three were determined to be at low risk for rejection, and one was reported as possibly at high risk (DFE >1%).

[0230] In conclusion, this study demonstrates that measuring dd-cfDNA in pregnant kidney recipients can distinguish between renal rejection and hypertensive disorders of pregnancy in a specific high-risk population of kidney transplant recipients. This noninvasive surrogate blood marker can help distinguish between acute rejection and hypertensive disorders of pregnancy. The presence of cfDNA in pregnant people demonstrates its potential as a diagnostic and monitoring tool for transplant rejection. Investigating the correlation between cfDNA levels and the occurrence of rejection in pregnant women who receive kidney transplants may improve risk assessment and patient management. This study could have a significant impact on the approximately 3,500 women of reproductive age who receive solid organ transplants in the United States. * * * *

Claims

1. A method for preparing a composition of amplified DNA derived from a biological sample of a maternal transplant recipient that is useful for determining the transplant status, (a) Extracting cell-free DNA (cfDNA) from the biological sample of the maternal transplant recipient, wherein the extracted cfDNA includes donor-derived cell-free DNA (dd-cfDNA) from the transplant, recipient-derived cell-free DNA (rd-cfDNA) from the maternal transplant recipient, and fetal-derived cell-free DNA (fd-cfDNA) from the fetus, (b) Preparing a composition in which a plurality of target loci are enriched from the cfDNA, wherein the target loci include one or more SNP loci in which the maternal transplant recipient and the biological father of the fetus are homozygous, in order to ensure homozygosity of the fetus at the SNP loci, so that the heterozygosity observed in the extracted cfDNA at the SNP loci originates from the transplant. (c) A method comprising: quantifying the amount of dd-cfDNA based on heterozygosity at the SNP locus; and determining whether the amount of dd-cfDNA or its function exceeds a cutoff threshold indicating transplant rejection.

2. The method according to claim 1, wherein quantifying the cfDNA and dd-cfDNA includes preparing a sequencing library from the extracted cfDNA and sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads.

3. The method according to claim 1, wherein the target gene loci comprise 10 to 50,000 target gene loci, and the method further comprises performing multiple target amplification of the DNA at the 10 to 50,000 target gene loci in a single reaction volume.

4. The method according to claim 1, further comprising step (c) determining the amount of transplant-derived alleles at one or more SNP loci and determining whether the amount or function thereof of the transplant-derived alleles at one or more SNP loci exceeds a cutoff threshold indicating transplant rejection, wherein transplant rejection is determined by a combination of (i) the amount or function thereof of the transplant-derived alleles at one or more SNP loci and (ii) the total amount or proportion of the dd-cfDNA.

5. A method for administering immunosuppressive therapy to a maternal transplant recipient, (a) Quantifying the total amount of cfDNA and the amount of dd-cfDNA in the biological sample of the transplant recipient according to the method of claim 1, (b) A method comprising titrating the dose of the immunosuppressive therapy according to the amount or function thereof of cfDNA and the amount or function thereof of dd-cfDNA.

6. The method according to claim 5, further comprising longitudinally repeating step (a) for the same transplant recipient, and determining the longitudinal changes in the amount or function thereof of cfDNA and the longitudinal changes in the amount or function thereof of dd-cfDNA.

7. The method according to claim 6, further comprising titrating the dose of immunosuppressive therapy in accordance with the longitudinal change in the total amount or function thereof of cfDNA and the longitudinal change in the amount or function thereof of dd-cfDNA.

8. Elevated levels of dd-cfDNA indicate transplant rejection and the need for adjustment of immunosuppressive therapy. The method according to claim 5, wherein a change or decrease in dd-cfDNA levels indicates transplant tolerance or stability and the need for adjustment of immunosuppressive therapy.

9. The method according to claim 1, further comprising sequencing the paternal DNA of the biological father of the fetus, and identifying one or more SNP loci in which the dd-cfDNA contains a heterozygous allele, while the maternal transplant recipient and the biological father of the fetus are homozygous.

10. The method according to claim 1, wherein the method is performed without prior knowledge of the donor and / or recipient's genotypes.

11. The method according to claim 1, further comprising performing universal amplification of the extracted DNA.

12. The method according to claim 11, wherein the universal amplification step preferentially amplifies dd-cfDNA over rd-cfDNA and fd-cfDNA.

13. The method according to claim 1, wherein the extraction step includes size selection to concentrate dd-cfDNA and reduce the amounts of rd-cfDNA and fd-cfDNA.

14. The method according to claim 1, wherein the amount of cfDNA is measured by quantitative PCR, real-time PCR, digital PCR, sequencing, microarray, or molecular barcoding and microscopic imaging.

15. The method according to claim 1, wherein the amount of dd-cfDNA is determined by using ratiometric and / or machine learning-artificial intelligence comparisons at one or more time points in time.

16. The method according to claim 1, wherein the cutoff threshold is the estimated ratio of dd-cfDNA to total cfDNA or a function thereof.

17. The method according to claim 1, wherein the amount of dd-cfDNA exceeding 1% of total cfDNA indicates that the transplant will cause acute rejection, and the amount of dd-cfDNA less than 1% of total cfDNA indicates that the transplant will cause borderline rejection, other damage, or be stable.

18. The method according to claim 1, wherein the transplant recipient has received one or more transplants selected from the following: kidney, liver, pancreas, intestine, heart, lung, heart / lung, stomach, testis, penis, ovary, uterus, thymus, face, hand, leg, bone, bone marrow, cornea, skin, pancreatic islet cells, heart valve, blood vessel, and blood transfusion.

19. The method according to claim 1, wherein the sample is obtained from the transplant recipient within 18 months after transplantation.

20. The method according to claim 1, wherein the risk of rejection of the transplant recipient is determined using logistic regression, random forest, or decision tree machine learning analysis.

21. The method according to claim 20, wherein the logistic regression, random forest, or decision tree machine learning analysis further incorporates one or more parameters selected from time after transplantation, age of transplant recipient and / or transplant donor, and sex of transplant recipient and / or transplant donor.

22. The method according to claim 1, wherein the biological sample is blood, serum, plasma, or urine.

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