Non-invasive method for assessing transplant rejection of pregnant transplant receptors

Abstract

The present disclosure provides methods for the preparation and analysis of a biological sample for maternal transplantation of a receptor wherein the method comprises extracting cell-free DNA from the receptor wherein the cell-free DNA comprises donor-derived cell-free DNA, receptor-derived cell-free DNA, and fetal-derived cell-free DNA, and measuring the amount of cell-free DNA and donor-derived cell-free DNA enables assessment of transplant rejection. The detection of the donor-derived cell-free DNA may be performed based on a locus at which the maternal transplantation receptor and the biological father of the fetus are homozygote and the transplantation donor is heterozygous.

Description

BACKGROUND OF THE INVENTION

[0001] Rapid detection of graft injury and / or rejection remains a challenge for transplant recipients. This challenge is exacerbated in pregnant kidney transplant recipients due to the common occurrence of renal dysfunction and chronic hypertension. In this population, it can be difficult to distinguish transplant rejection, renal disease progression, and preeclampsia in a non-invasive manner.

[0002] After transplantation, tests for determining and monitoring graft injury and / or rejection are a key aspect of post-transplant care and the determination of personalized immunosuppressive therapy. In addition to being dangerous to the mother and fetus, traditional biopsy-based tests are invasive and costly, and may result in late diagnosis of graft injury and / or rejection. Accordingly, there is a need for a non-invasive test for graft injury and / or rejection that enables early diagnosis of graft injury and / or rejection in pregnant transplant recipients, and that is safer, more sensitive, and more specific than traditional biopsy-based tests.

[0003] Accordingly, there is a need for improved methods for accurately diagnosing, screening, testing, and monitoring graft injury and / or rejection in pregnant transplant recipients at an early stage. The present disclosure meets this need. SUMMARY OF THE INVENTION

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

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

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

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

[0008] In one aspect, the present disclosure relates to a method for preparing a DNA composition from a biological sample of a maternal transplant recipient for determining transplant rejection, wherein the DNA composition comprises one or more target loci comprising alleles of transplant origin; and wherein the determining step further includes determining the amount of alleles of transplant origin at the one or more SNP loci and determining whether the amount or a function of the alleles of transplant origin at the one or more SNP loci exceeds a cut-off threshold indicative of transplant rejection; wherein transplant rejection is determined by a combination of (i) the amount or a function of the alleles of transplant origin at the one or more target loci and (ii) the total amount or fraction of dd-cfDNA.

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

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

[0011] In some embodiments of the methods described herein, the selected target loci comprise 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, the extracted DNA is subjected to universal amplification. In some embodiments, universal amplification preferentially amplifies dd-cfDNA rather than rd-cfDNA and fd-cfDNA.

[0014] In some embodiments of the methods described herein, extracting cfDNA from a biological sample includes size selection to enrich dd-cfDNA and reduce the amounts 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 microscopy imaging (such as NanoString ). In some embodiments of the methods described herein, the amount of dd-cfDNA is determined using ratio-based and / or machine learning-artificial intelligence comparisons at single or multiple time points.

[0016] In some embodiments of the methods described herein, the cut-off threshold is the estimated percentage of dd-cfDNA in 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 is undergoing acute rejection, and wherein an amount of dd-cfDNA less than 1% of total cfDNA indicates that the transplant is undergoing critical rejection, is undergoing other injury, or is stable.

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

[0018] In some embodiments of the methods described herein, logistic regression, random forest, or decision tree machine learning analysis is used to determine the rejection risk of the transplant recipient. 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 the group consisting of: time post-transplant, age of the transplant recipient and / or transplant donor, sex 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 DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . Workflow for detecting donor-derived cell-free DNA in human plasma samples from pregnant transplant recipients.

[0021] Figure 2. Workflow for measuring the dd-cfDNA fraction in pregnant kidney transplant recipients (KTRs).

[0022] Figure 3. Illustration representing the dd-cfDNA test and the medical history of two pregnant KTRs.

[0023] Figure 4. Heterozygosity plots of two pregnant KTRs with (a) low and (b) high DFE.

[0024] Figure 5. Measurement of donor-derived fraction in pregnant women with KTRs. DETAILED DESCRIPTION

[0025] 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), which is incorporated herein by reference in its entirety.

[0026] WO2020 / 010255, titled “Methods for Detection of Donor-Derived Cell-Free DNA”, filed on Jul. 3, 2019 as PCT / US2019 / 040603, is incorporated herein by reference in its entirety.

[0027] WO2021 / 243045, titled “Methods for Detection of Donor-Derived Cell-Free DNA”, filed on May 27, 2021 as PCT / US2021 / 034561, is incorporated herein by reference in its entirety.

[0028] The present disclosure relates to methods for determining and monitoring transplant rejection in a pregnant recipient based on targeted enrichment and high-throughput sequencing of cell-free DNA from a biological sample 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 presented herein illustrate that the methods of the present disclosure can be used to detect donor-derived nucleic acids in biological samples collected from a pregnant transplant recipient.

[0029] Methods for determining and monitoring transplant rejection based on measuring cell-free DNA.

[0030] In one aspect, the present disclosure relates to a method of preparing a DNA composition derived from a biological sample of a maternal transplant recipient for determining a transplant status, the method comprising:

[0031] (a) Extract cell-free DNA (cfDNA) from the biological sample of the maternal transplant recipient, wherein the extracted cfDNA comprises 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;

[0032] (b) Prepare a composition from the cfDNA, wherein one or more selected target loci are enriched, 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 the heterozygosity observed at the selected target loci in the extracted cfDNA is derived from the transplant; and

[0033] (c) Quantify the amount of the extracted cfDNA and the amount of dd-cfDNA based on the heterozygosity at the selected target loci to determine whether the amount of dd-cfDNA or a function thereof exceeds a cut-off threshold indicative of transplant rejection. In some embodiments, the method further comprises preparing a sequencing library from the extracted cfDNA and sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads. In some embodiments, the extracted cfDNA is not amplified or pre-amplified prior to sequencing. In some embodiments, the selected target loci comprise 10 - 50,000 target loci, and the method further comprises performing multiplex targeted amplification of the DNA at the 10 - 50,000 target loci in a single reaction volume.

[0034] In one aspect, the present disclosure relates to a method for preparing a DNA composition, the DNA being derived from a biological sample of a maternal transplant recipient, the method for determining a transplant status, the method comprising:

[0035] (a) Extract cfDNA from the biological sample of the maternal transplant recipient, wherein the extracted cfDNA comprises donor-derived cell-free DNA (dd-cfDNA), recipient-derived cell-free DNA (rd-cfDNA), and fetal-derived cell-free DNA (fd-cfDNA);

[0036] (b) Prepare a sequencing library from the extracted cfDNA, sequence the sequencing library by high-throughput sequencing to obtain sequencing reads, and quantify the total amount of dd-cfDNA based on the sequencing reads;

[0037] (c) Determine whether the amount of dd-cfDNA or a function thereof exceeds a cut-off threshold indicative of transplant rejection or injury. In some embodiments, the extracted cfDNA is not amplified or pre-amplified prior to sequencing. In some embodiments, prior to sequencing, the extracted cfDNA is multiplexed and targeted amplified at 10 - 50,000 target loci in a single reaction volume. In some embodiments, preparing a sequencing library includes attaching adapters to the extracted cfDNA, such as by ligation. In some embodiments, attaching adapters to the extracted cfDNA includes end repair, adding adenosine to the cfDNA fragments, and subsequent sticky-end ligation to the cfDNA fragments. In some embodiments, the cfDNA fragments are repaired and filled in to generate blunt ends. In some embodiments, attaching adapters to the extracted cfDNA includes blunt-end ligation of the adapters to the cfDNA fragments. In some embodiments, the adapters are attached during the amplification step.

[0038] In one aspect, the present disclosure relates to a method of preparing an amplified DNA composition, the DNA being derived from a biological sample of a maternal transplant recipient, the method for determining a transplant status, the method comprising:

[0039] (a) Extract cfDNA from the biological sample of the maternal transplant recipient, wherein the extracted cfDNA comprises dd-cfDNA, rd-cfDNA, and cell-free DNA of fetal origin from the transplant;

[0040] (b) Prepare an amplified DNA composition by multiplexed and targeted amplifying the extracted cfDNA at 10 - 50,000 target loci in a single reaction volume to detect and quantify the amount of dd-cfDNA; and

[0041] (c) Determine whether the amount of dd-cfDNA or a function thereof exceeds a cut-off threshold indicative of transplant rejection.

[0042] In one aspect, the present disclosure relates to a method of preparing an amplified DNA composition, the DNA being derived from a biological sample of a maternal transplant recipient, the method for determining a transplant status, the method comprising:

[0043] (a) Extract cfDNA from the blood of the maternal transplant recipient, wherein the extracted cfDNA comprises dd-cfDNA, rd-cfDNA, and fd-cfDNA;

[0044] (b) Preparing an amplified DNA composition by targeted amplification of the extracted cfDNA at 10 - 50,000 target loci in a single reaction volume, sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amounts of dd-cfDNA, transplanted rd-cfDNA, and fd-cfDNA based on the sequencing reads, wherein the donor loci, recipient loci, and fetal loci are the same, and wherein donor reads, recipient reads, and fetal reads are distinguished based on the inserted sequences;

[0045] (c) Determining whether the fraction of dd-cfDNA or a function thereof exceeds a cut-off threshold indicative of transplant rejection. As used herein, an "inserted sequence" refers to any sequence that is different in the transplant recipient target locus compared to the same transplant donor target locus.

[0046] In one aspect, the present disclosure relates to a method for preparing an amplified DNA composition, the DNA being derived from a biological sample of a maternal transplant recipient, the method for determining transplant status, the method comprising:

[0047] (a) Extracting cfDNA from the biological sample of the maternal transplant recipient, wherein the extracted cfDNA comprises dd-cfDNA, rd-cfDNA, and fd-cfDNA;

[0048] (b) Preparing an amplified DNA composition by targeted amplification of the extracted cfDNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise one or more target loci indicative of transplant rejection; and

[0049] (c) Determining the amount of the one or more target loci indicative of transplant rejection, and determining whether the amount of the one or more target loci indicative of transplant rejection or a function thereof exceeds a cut-off threshold indicative of transplant rejection.

[0050] In one aspect, the present disclosure relates to a method for preparing a DNA composition, the DNA being derived from a biological sample of a maternal transplant recipient, the method for determining transplant status, wherein the DNA composition comprises one or more target loci indicative of transplant rejection; and wherein the determining step further comprises determining the amount of the one or more target loci indicative of transplant rejection, and determining whether the amount of the one or more target loci indicative of transplant rejection or a function thereof exceeds a cut-off threshold indicative of transplant rejection; wherein transplant rejection is determined by a combination of (i) the amount of the one or more target loci indicative of transplant rejection or a function thereof and (ii) the total amount of dd-cfDNA or the fraction of dd-cfDNA.

[0051] In one aspect, the present disclosure relates to a method of administering an immunosuppressive therapy in a maternal transplant recipient, the method comprising:

[0052] (a) Quantifying the total amount of cfDNA and the amount of dd-cfDNA in a biological sample of the transplant recipient according to any of the methods disclosed herein; and

[0053] (b) Titrating the dose 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 longitudinally repeating step (a) for the same transplant recipient, and determining the longitudinal change in the amount of cfDNA or a function thereof and the longitudinal change in the amount of dd-cfDNA or a function thereof.

[0054] 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 the maternal transplant recipient.

[0055] Sample comprising nucleic acids and method for obtaining sample and extracting nucleic acids

[0056] 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 human. In some embodiments, the transplant is from a pig, primate, baboon, cow or dog. In some embodiments, the transplant can be an allograft or a xenograft. In some embodiments, the transplant can be an organ transplant, a tissue transplant, a cell transplant or a fluid transplant.

[0057] In some embodiments, the transplant recipient has received a plurality of 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, islet cells, heart valve and blood vessel. In some embodiments, one or more of the transplanted organs are from the same transplant donor. In some embodiments, one or more of the transplanted organs are from a plurality of different transplant donors. In some embodiments, the transplant recipient has received a simultaneous transplant of more than one organ. 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 donor different from the organ donor.

[0058] In some embodiments, the transplant recipient has received a transplanted organ selected from one or more of the following: 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.

[0059] In some embodiments, the sample is obtained from the transplant recipient less than 18 months after transplantation, less than 17 months after transplantation, less than 16 months after transplantation, less than 15 months after transplantation, less than 14 months after transplantation, less than 13 months after transplantation, or less than 12 months after transplantation. In some embodiments, the sample is obtained from the transplant recipient between 0 and 2 months after transplantation, between 2 and 4 months after transplantation, between 4 and 6 months after transplantation, between 6 and 9 months after transplantation, between 9 and 12 months after transplantation, or between 12 and 18 months after transplantation.

[0060] 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 pregnant for less than 3 months. In some embodiments, the transplant recipient is pregnant for less than six months. In some embodiments, the transplant recipient is pregnant for less than nine months. In some embodiments, the transplant recipient is pregnant for more than nine months. In some embodiments, the transplant recipient has just given birth. In some embodiments, the transplant recipient gave birth less than one day ago, i.e., less than one day postpartum. In some embodiments, the transplant recipient gave birth less than one week ago, i.e., less than one week postpartum. In some embodiments, the transplant recipient gave birth less than one month ago, i.e., less than one month postpartum. In some embodiments, the transplant recipient gave birth less than three months ago, i.e., less than three months postpartum. In some embodiments, the transplant recipient gave birth less than one year ago, 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.

[0061] 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 the 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.

[0062] 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 a simultaneous transplant of more than one organ from one or more different donors.

[0063] The biological sample can be a body fluid sample, tissue, organ, or single cell. In some embodiments, the biological sample includes 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 can be extracellular vesicles derived from a body fluid sample such as blood, plasma, serum, CSF, or urine.

[0064] Nucleic acids and methods for extracting or enriching nucleic acids

[0065] The methods disclosed herein include extracting nucleic acids from a sample derived from a subject. In some embodiments, the subject can be a pregnant transplant recipient. In some embodiments, the subject can be the father of the transplant recipient's fetus. In some embodiments, the subject can be the transplant donor. In some embodiments, the subject can be a fetus. The nucleic acid can be genomic DNA, cDNA, cell-free DNA (cfDNA), cell-free mitochondrial DNA (cf mDNA), cell-free DNA derived from nuclear DNA (cfnDNA), cellular DNA, or mitochondrial DNA. CfDNA can be derived from exosomes or microvesicles. In some embodiments, the nucleic acid can 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 can be cell-free RNA, cellular RNA, or exosomal RNA. In some embodiments, the RNA includes small non-coding RNA (sncRNA). In some embodiments, the sncRNA includes microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), or miscellaneous RNA (miscRNA). In some embodiments, the cell-free sncRNA is derived from exosomes or microvesicles.

[0066] In some embodiments, nucleic acids are extracted by using size exclusion. In some embodiments, cfDNA is separated from cellular DNA based on size. In some embodiments, nucleic acids are separated by using magnetic or otherwise labeled affinity chromatography.

[0067] In some embodiments, nucleic acids are preferentially enriched. Nucleic acids are preferentially enriched by using preferential enrichment at a locus or target site. Such preferential enrichment refers to any method that results in a higher percentage of nucleic acid molecules corresponding to the locus in the nucleic acid mixture after enrichment than in the nucleic acid mixture before enrichment. The method can involve selectively amplifying nucleic acid molecules corresponding to the locus. The method can involve removing nucleic acid molecules that do not correspond to the locus. The method can involve a combination of methods. The enrichment level is defined as the percentage of nucleic acid molecules corresponding to the locus or target in the mixture after enrichment divided by the percentage of nucleic acid molecules corresponding to the locus or target in the mixture before enrichment. Preferential enrichment can be performed at multiple loci. In some embodiments of the present disclosure, the enrichment level is greater than 20. In some embodiments of the present disclosure, the enrichment level is greater than 200. In some embodiments of the present disclosure, the enrichment level is greater than 2,000. When preferential enrichment is performed at multiple loci, the enrichment level can refer to the average enrichment level of all loci in the locus group.

[0068] Amplification refers to a method of increasing the copy number of nucleic acid molecules. Selective amplification can refer to a method of increasing the copy number of specific nucleic acid molecules or nucleic acid molecules corresponding to a specific nucleic acid molecule region. It can also refer to a method that increases the copy number of specific targeted nucleic acid molecules or targeted nucleic acid molecule regions more than that of non-targeted nucleic acid molecules or regions.

[0069] Selective amplification can be a method of preferential enrichment. A universal primer sequence refers to a DNA sequence that can be attached to a population of target DNA molecules, for example, by ligation, PCR, or ligation-mediated PCR. Once added to the target molecule population, a universal primer sequence-specific primer can be used to amplify the target population using a single pair of amplification primers. The universal primer sequence is generally not related to the target sequence. A universal adapter or ligation adapter or library tag is a DNA molecule containing a universal primer sequence that can be covalently linked to the 5'-major end and 3'-major end of a population of target double-stranded DNA molecules. The addition of the adapter provides universal primer sequences at the 5'-major end and 3'-major end of the target population from which PCR amplification can be performed to amplify all molecules of the target population using a single pair of amplification primers. Targeting refers to a method for selectively amplifying or otherwise preferentially enriching DNA molecules that correspond to a set of loci in a DNA mixture.

[0070] Specific nucleic acids can also be enriched by using hybridization capture. In some embodiments, preferentially enriching DNA at multiple loci or target sites includes: obtaining a set of hybridization capture probes; hybridizing the hybridization capture probes with the DNA in the sample; and physically separating the hybridized DNA from the unhybridized DNA in the DNA sample.

[0071] In some embodiments, in the methods disclosed herein, DNA is preferentially enriched at target gene loci or biomarkers.

[0072] The term "biomarker" refers to a molecule that is an indicator species of an abnormal biological condition (e.g., a disease or disorder or transplant rejection). For example, a biomarker can be a gene or gene product (i.e., RNA or protein) that (a) is expressed at a higher or lower level, (b) has an altered ratio relative to another biomarker, (c) is present at a higher or lower level, (d) is a variant or mutant of a gene product, or (e) is simply present or absent in a cell or tissue sample from a subject with or suspected of having a disease compared to an unaffected tissue or cell sample from a subject with or suspected of having a disease or a group of subjects without or suspected of having a disease. In the context of transplantation, a biomarker can indicate poor donor organ health or transplant rejection. That is, one or more gene products are specific enough for a test sample such that one or more can be used to identify, predict, or detect the presence of transplant rejection, a disease, disease risk, risk of a given event, or a change in disease state, or to inform an appropriate or improved treatment regimen. In the methods described herein, dd-cfDNA levels above a threshold can be considered biomarkers.

[0073] In some embodiments, one or more biomarkers can be one or a collection of genetic aberrations, which are used herein to refer to the amount of nucleic acid and nucleic acid variants within a particle containing nucleic acid. Specifically, genetic aberrations include, but are not limited to, overexpression of a gene (e.g., an oncogene) or genome, underexpression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or genome, alternative production of splice variants of a gene or genome, gene copy number variants (CNVs) (e.g., DNA double minutes), nucleic acid modifications (e.g., methylation, acetylation, and phosphorylation), single nucleotide polymorphisms (SNPs), chromosomal rearrangements (e.g., inversions, deletions, and duplications), and mutations (insertions, deletions, duplications, missense, nonsense, synonymous, or any other nucleotide change) of a gene or genome, which in many cases ultimately affect the activity and function of the gene product, resulting in alternative transcriptional splice variants and / or changes in gene expression levels, or a combination of any of the foregoing.

[0074] In some embodiments, preferentially enriching DNA at multiple polymorphic loci includes: obtaining a plurality of pre-circularized probes, wherein each probe targets one of the polymorphic loci in the polymorphic loci, and wherein the 3' and 5' ends of the probe are designed to hybridize to a DNA region that is separated from the polymorphic locus of the locus by a small number of bases, wherein 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 to 25, 26 to 30, 31 to 60, or a combination thereof; hybridizing the pre-circularized probes to DNA from a sample; filling the gap between the ends of the hybridized probes using a DNA polymerase; circularizing the pre-circularized probes; and amplifying the circularized probes.

[0075] In some embodiments, preferentially enriching DNA at multiple polymorphic loci includes: obtaining a plurality of ligation-mediated PCR probes, wherein each PCR probe targets one of the polymorphic loci in the polymorphic loci, and wherein the upstream and downstream PCR probes are designed to hybridize to a DNA region on one DNA strand that is separated from the polymorphic locus of the locus by a small number of bases, wherein 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 to 25, 26 to 30, 31 to 60, or a combination thereof; hybridizing the ligation-mediated PCR probes to DNA from a first sample; filling the gap between the ends of the ligation-mediated PCR probes using a DNA polymerase; ligating the ligation-mediated PCR probes; and amplifying the ligated ligation-mediated PCR probes.

[0076] In some embodiments, preferentially enriching DNA at multiple polymorphic loci includes: obtaining a plurality of hybridization capture probes that target the polymorphic loci; hybridizing the hybridization capture probes to the DNA in the sample; and physically removing some or all of the unhybridized DNA from the first DNA sample.

[0077] In some embodiments, the hybridization capture probes are designed to hybridize to regions flanking but not overlapping with the polymorphic locus. In some embodiments, the hybridization capture probes are designed to hybridize to regions flanking but not overlapping with the polymorphic locus, and wherein the length of the flanking capture probes 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 hybridization capture probes are designed to hybridize to regions overlapping with the polymorphic locus, and wherein a plurality of hybridization capture probes includes at least two hybridization capture probes for each polymorphic locus, and wherein each hybridization capture probe is designed to be complementary to a different allele at the polymorphic locus.

[0078] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci comprises: obtaining a plurality of internal forward primers, wherein each primer targets one of the polymorphic loci in the polymorphic locus, and wherein the 3' end of the internal forward primer is designed to hybridize to a DNA region upstream of the polymorphic locus and is separated from the polymorphic locus by a small number of bases, wherein the small number 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; optionally obtaining a plurality of internal reverse primers, wherein each primer targets one of the polymorphic loci in the polymorphic locus, and wherein the 3' end of the internal reverse primer is designed to hybridize to a DNA region upstream of the polymorphic locus and is separated from the polymorphic locus by a small number of bases, wherein the small number 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 internal primers to the DNA; and amplifying the DNA using polymerase chain reaction to form amplicons.

[0079] In some embodiments, the method further comprises: obtaining a plurality of external forward primers, wherein each primer targets one of the polymorphic loci in the polymorphic locus, and wherein the external forward primers are designed to hybridize to a DNA region upstream of the internal forward primers; optionally obtaining a plurality of external reverse primers, wherein each primer targets one of the polymorphic loci in the polymorphic locus, and wherein the external reverse primers are designed to hybridize to a DNA region immediately downstream of the internal reverse primers; hybridizing the first primers to the DNA; and amplifying the DNA using polymerase chain reaction.

[0080] In some embodiments, the method further comprises: obtaining a plurality of external reverse primers, wherein each primer targets one of the polymorphic loci, and wherein the external reverse primers are designed to hybridize to a DNA region located immediately downstream of the internal reverse primer; optionally obtaining a plurality of external forward primers, wherein each primer targets one of the polymorphic loci, and wherein the external forward primers are designed to hybridize to a DNA region located upstream of the internal forward primer; hybridizing a first primer to the DNA; and amplifying the DNA using polymerase chain reaction.

[0081] In some embodiments, preparing the sample comprises incorporating a universal adaptor into the DNA and amplifying the DNA using polymerase chain reaction. In some embodiments, at least a portion of the amplified amplicons is 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 fraction is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%.

[0082] In some embodiments, amplifying the DNA is performed in one or more separate reaction volumes, and wherein 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.

[0083] In some embodiments, preparing the sample further comprises dividing the sample into a plurality of portions, and wherein the DNA in each portion is preferably enriched at a subset of the plurality of polymorphic loci. In some embodiments, the internal primers are selected by identifying primer pairs that may form undesired primer duplexes and removing at least one primer pair identified as likely to form an undesired primer duplex from the plurality of primers. In some embodiments, the internal primers contain regions designed to hybridize upstream or downstream of the targeted polymorphic locus and optionally contain a universal priming sequence designed to allow PCR amplification. In some embodiments, at least some of the primers further contain a random region that is different for each individual primer molecule. In some embodiments, at least some of the primers further contain a molecular barcode.

[0084] In some embodiments, the method includes: (a) performing multiplex polymerase chain reaction (PCR) on a nucleic acid sample comprising a target locus in a single reaction volume using (i) at least 10 different primer pairs or (ii) at least 10 target-specific primers and universal or tag-specific primer pairs to simultaneously amplify at least 10 different target loci, thereby generating an amplification product comprising target amplicons; and (b) sequencing the amplification product. In some embodiments, the method does not include the use of a microarray.

[0085] In some embodiments, the method includes: (a) performing multiplex polymerase chain reaction (PCR) on a cfDNA sample comprising a target locus in a single reaction volume using (i) at least 10 different primer pairs or (ii) at least 10 target-specific primers and universal or tag-specific primer pairs to simultaneously amplify at least 10 different target loci, thereby generating an amplification product comprising target amplicons; and (b) sequencing the amplification product. In some embodiments, the method does not include the use of a microarray.

[0086] After blood draw and prior to nucleic acid extraction, blood cells within the blood sample may burst and shed long DNA fragments into the sample, which will increase the total amount of cfDNA and background noise, thereby distorting the percentage of detected dd-cfDNA. To reduce such background noise, and based on the observation that dd-cfDNA is generally shorter than DNA fragmented from transplant recipient blood cells, two specific enrichments of dd-cfDNA were considered. In one embodiment, size selection is applied to select shorter cfDNA. In another embodiment, based on the hypothesis that shorter dd-cfDNA (usually in the form of mononucleosomes) is amplified more efficiently than longer transplant recipient-derived DNA, a universal amplification step is applied to reduce the noise (e.g., prior to applying multiplex PCR).

[0087] Target genes and loci

[0088] In some embodiments, the methods disclosed herein include selecting target loci that are homozygous in both the maternal transplant recipient and the biological father of the fetus, thereby ensuring homozygosity of the fetus. Thus, any heterozygosity at the loci detected in the extracted cfDNA will be from the donor. In some embodiments, the detected heterozygosity in the extracted cfDNA can be used to quantify the amount of dd-cfDNA present in the biological sample.

[0089] In some embodiments, the methods disclosed herein further include preferentially enriching cfDNA at a plurality of target loci selected based on homozygosity of both the maternal transplant recipient and the biological father of the fetus. In some embodiments, 10 to 50,000 target loci are selected and enriched.

[0090] In some embodiments, the methods disclosed herein further include sequencing the paternal genome of the fetus in order to select target loci in 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.

[0091] The nucleic acid can include biomarkers indicative of an immune response or various diseases or conditions as 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 recipient target locus set, a fetal target locus set, and a donor target locus set, wherein each of the recipient target locus set, the fetal target locus set, and the donor target locus set is different. In some embodiments, the recipient locus set and the fetal target locus set are the same, and the donor target locus set is different. In some embodiments, each of the recipient target locus set, the fetal target locus set, and the donor target locus set is the same, and one or more recipient target loci and / or fetal target loci can be distinguished from the corresponding donor loci by an inserted sequence. As used herein, an "inserted sequence" refers to any sequence that is different in the target loci of the transplant recipient and / or the fetus compared to the same transplant donor target locus.

[0092] In some embodiments, the method includes extracting fragmented or intact cfDNA from a sample of the transplant recipient, wherein the extracted cfDNA comprises cfDNA of donor, fetal, and / or recipient origin, and wherein the cfDNA comprises multiple biomarkers indicative of an immune response or a disease or disorder. In some embodiments, the biomarker indicates an increased immune response. In some embodiments, the biomarker indicates a decreased immune response.

[0093] In some embodiments, the methods of the present disclosure include preselecting cfDNA target molecules. In some embodiments, the cfDNA target molecules comprise cfDNA species known to be associated with assessing organ health. In some embodiments, the present disclosure provides methods for identifying cfDNA target molecules associated with assessing organ health.

[0094] In some embodiments, the methods disclosed herein further include preferentially enriching cfDNA at multiple target loci or biomarkers indicative of transplant rejection. In some embodiments, the cfDNA biomarkers indicate an increased immune response or a decreased immune response.

[0095] In some embodiments, the target loci and / or biomarkers comprise single nucleotide polymorphism (SNP) loci. Samples and methods for isolating nucleic acids from samples

[0096] In some embodiments, the nucleic acid sample comprises fragmented or digested nucleic acid. In some embodiments, the nucleic acid sample comprises 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.

[0097] In some embodiments, the nucleic acid sample comprises 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 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 secretion, dental plaque, gastrointestinal tissue, feces, urine, hair, bone, body fluid, tears, tissue, skin, nail, blastomere, embryo, amniotic fluid, chorionic villus sample, bile, lymph, cervical mucus, or forensic sample. In some embodiments, the target locus is a segment of nucleic acid. In some embodiments, the target locus is a segment of nucleic acid present in the 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.

[0098] In some embodiments, the method comprises isolating or purifying DNA. A variety of standard procedures are known in the art for achieving such purposes. In some embodiments, the sample can be centrifuged to separate the layers. In some embodiments, filtration can be used to isolate DNA. In some embodiments, the preparation of DNA can involve amplification, fractionation, purification by chromatography, liquid fractionation, isolation, preferential enrichment, preferential amplification, targeted amplification, or any of a variety of other techniques known in the art or described herein. In some embodiments of isolating DNA, RNase is used to degrade RNA. In some embodiments, QIAamp TM DNA Mini Kit (Qiagen) is used to isolate DNA according to the manufacturer's protocol. In some embodiments, MagMAX TM Cell-Free DNA Isolation Kit (Applied Biosystems) is used to isolate cfDNA molecules. Nanovue (GE Healthcare, Piscataway, N.J., USA) can optionally be used to determine the concentration and purity of DNA, and the DNA integrity can optionally be measured by using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA).

[0099] In some embodiments, adapters are added to construct a sequencing library. Prior to ligation, the sample DNA can be blunt-ended and then a single adenosine base can be added to the 3'-end. In some embodiments, the ligation of the adapter to the nucleic acid is a sticky-end ligation. Prior to ligation, the DNA can be cleaved using a restriction enzyme or some other cleavage method. During ligation, the 3'-adenosine of the sample fragment and the complementary 3'-tyrosine overhang of the adapter can enhance the ligation efficiency. In some embodiments, an adapter ligation kit present in the AGILENT SURESELECT TM kit is used for adapter ligation. In some embodiments, the adapter is incorporated into the sequence by PCR. In some embodiments, the adapter is incorporated into the sequence during universal or targeted amplification.

[0100] In some embodiments, universal primers are used to amplify the library. In one embodiment, the amplified library is fractionated by size selection or by using products such as AGENCOURT AMPURE TM beads or other similar methods. In some embodiments, PCR amplification is used to amplify target loci. In some embodiments, the amplified DNA is sequenced (such as using an ILLUMINAIIGAX TM or HiSeq sequencer). In some embodiments, the amplified DNA is sequenced from each end of the amplified DNA to reduce sequencing errors. If there is a sequence error in a particular base when sequencing from one end of the amplified DNA, it is less likely that there will be a sequence error in the complementary base when sequencing from the other side of the amplified DNA (compared to sequencing multiple times from the same end of the amplified DNA).

[0101] As a non-limiting example, a locus can be a single nucleotide polymorphism (SNP), an intron, or an exon. In some embodiments, a locus can include an insertion, deletion, or translocation. In some embodiments, the sample can include a blood, serum, or plasma sample. In some embodiments, the sample can include cell-free DNA in a blood, serum, or plasma sample (such as circulating cell-free tumor DNA, circulating cell-free donor DNA, or circulating cell-free fetal DNA). In these embodiments, the sample typically comes from an animal, such as a mammal or a human, and is typically present in fragments of about 160 nucleotides in length. In some embodiments, after removing cell debris and platelets by centrifugation, EDTA-2Na tubes are used to isolate cell-free DNA from blood. Plasma samples can be stored at -80 °C until use, for example, with QIAamp TMDNA was extracted using a DNA Mini Kit (Qiagen, Hilden, Germany) (e.g., Hamakawa et al., Br J Cancer. 2015;112:352-356).

[0102] Many kits and methods for generating nucleic acid molecule libraries for subsequent sequencing are known in the art. Kits that are particularly suitable for preparing libraries from small nucleic acid fragments, especially circulating cell-free DNA, can be used to practice the methods provided herein. For example, the NEXTflex TM Cell Free Kit (Bioo Scientific, Austin, Tex.) or the Natera Library Prep Kit (Natera, San Carlos, Calif.). Such kits are typically modified to include adapters customized for the amplification and sequencing steps in the methods provided herein. Commercially available kits (such as the ligation kit present in the Agilent SureSelect TM Kit (Agilent, Santa Clara, Calif.)) can also be used for adapter ligation.

[0103] The sample nucleic acid molecule is composed of naturally occurring or non-naturally occurring ribonucleotides or deoxyribonucleotides linked by phosphodiester bonds. In addition, the sample nucleic acid molecule is composed of nucleic acid segments targeted for sequencing. The sample nucleic acid molecule can be or can include a nucleic acid segment having a length of at least 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides. In any of the embodiments disclosed herein, the lower end of the length range of the sample nucleic acid molecule or nucleic acid segment 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, while the upper end of the length range can be 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 the lower end of the length range of the nucleic acid molecule 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, while the upper end of the length range can be 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. For clarity, the nucleic acid initially isolated from a tissue, fluid, or cultured cell may be much longer than the sample nucleic acid molecule processed using the methods herein. As discussed herein, for example, such initially isolated nucleic acid molecules can be fragmented to generate nucleic acid segments prior to being used in the methods herein. In some embodiments, the nucleic acid molecule and the nucleic acid segment can be the same. The sample nucleic acid molecule or sample nucleic acid segment can include a target locus containing one or more nucleotides of interest, particularly single nucleotide polymorphisms or single nucleotide variants.In any of the disclosed embodiments, the length of 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, and the target locus can include a part or all of the sample nucleic acid molecule and / or the sample nucleic acid segment. In other embodiments, the lower end of the length range of the target locus 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, while the upper end of the length range can be 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 target loci on 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, the target loci on 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.

[0104] In some embodiments, the entire sample nucleic acid molecule is the sample nucleic acid segment. For example, in certain embodiments, wherein the adapter is directly ligated to the end of the sample nucleic acid molecule, or ligated to a nucleic acid that is ligated to the end of the sample nucleotide molecule, or ligated as part of a primer that binds to a sequence at the end of the sample nucleic acid segment, or ligated as an adapter (such as a universal adapter added thereto), as further discussed herein, the entire nucleic acid molecule can be the sample nucleic acid segment. In other embodiments, for example, certain embodiments, wherein the adapter is attached to the sample nucleic acid molecule as part of a primer that targets a binding site located within the end of the sample nucleic acid molecule, a part of the sample nucleic acid molecule can be the sample nucleic acid segment 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 the nucleic acid segment.

[0105] In some embodiments, the sample nucleic acid molecules are a mixture of nucleic acids isolated from a natural source, some of the sample nucleic acid molecules have the same sequence, some have sequences sharing at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity, and some have less than 50%, 40%, 30%, 20%, 10% or 5% sequence identity. The lower end of the entire range of the sample nucleic acid molecules is 20, 25, 50, 75, 100, 125, 150, 200, 250 nucleotides, and the upper end of the entire range is 50, 75, 100, 125, 150, 200, 250, 300, 400 or 500 nucleotides. Such sample nucleic acid molecules can be nucleic acid samples isolated from tissues or fluids of a mammal (such as a human) without enriching one sequence over another. In other embodiments, the target sequences (e.g., those from genes of interest) can be enriched prior to performing the methods provided herein.

[0106] Differentiating dd-cfDNA from fd-cfDNA

[0107] In some embodiments, dd-cfDNA is differentiated from fd-cfDNA based on the parental genotype. For example, in some embodiments, the paternal DNA from the biological father of the fetus is genotyped using the methods described herein. In some embodiments, the paternal genotype is already known. Based on the paternal and maternal transplant recipient genotypes, target loci that are homozygous in both the maternal and paternal genomes are selected, and thus the target loci will be homozygous in the fetal genome. Thus, the detection of heterozygosity at the target locus in cfDNA will be derived from transplantation and can be used to quantify the amount of dd-cfDNA in a biological sample.

[0108] Determining the rejection risk of a transplant recipient

[0109] In some embodiments, logistic regression, random forest, or decision tree machine learning analysis is used to determine the rejection risk of a transplant recipient. In some embodiments, the machine learning analysis incorporates the amount of dd-cfDNA or a function thereof in the sample of the transplant recipient as a parameter. In some embodiments, the machine learning analysis incorporates the number of reads of dd-cfDNA or a function thereof as a parameter. In some embodiments, the machine learning analysis incorporates the estimated percentage of dd-cfDNA in the total cfDNA as a parameter. In some embodiments, the machine learning analysis incorporates the amount of dd-cfDNA, the number of reads of dd-cfDNA, or the estimated percentage of dd-cfDNA in the total cfDNA in the sample of the transplant recipient as a parameter.

[0110] Machine learning can be used to solve the problem of rejection and non-rejection. Machine learning is disclosed in WO2020 / 018522, entitled "Methods and Systems for calling Ploidy States using a Neural Network", filed as PCT / US2019 / 041981 on July 16, 2019, which is incorporated herein by reference in its entirety. In some embodiments, the cut-off threshold is scaled according to the amount of total cfDNA in the biological sample.

[0111] In some embodiments, the cut-off threshold is expressed as the percentage of dd-cfDNA in the sample (dd-cfDNA %). In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of dd-cfDNA. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of dd-cfDNA per unit volume of the blood sample. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of dd-cfDNA per unit volume of the blood sample multiplied by the body weight, BMI or blood volume of the transplant recipient.

[0112] In some embodiments, the cut-off threshold takes into account the patient's body weight, BMI or blood volume. In some embodiments, the cut-off threshold takes into account one or more of the following: donor genomic copies / plasma volume, fetal genomic copies / plasma volume, number of fetuses present, pregnancy stage, cfDNA yield / plasma volume, donor height, donor weight, donor age, donor sex, donor race, donor organ mass, donor organ, living donor and deceased donor, family relationship (or lack thereof) between donor and recipient, recipient height, recipient weight, recipient age, recipient sex, recipient race, creatinine, eGFR (estimated glomerular filtration rate), cfDNA methylation, DSA (donor-specific antibody), KDPI (kidney donor profile index), drugs (immunosuppressants, steroids, blood thinners, etc.), infections (BKV, EBV, CMV, UTI), recipient, fetal and / or donor HLA alleles or epitope mismatches, Banff classification of kidney transplant pathology, and cause and surveillance or protocol biopsies.

[0113] In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 50% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 60% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 70% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 75% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 80% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 85% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 90% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled or adjusted according to the amount of total cfDNA in the biological sample and a 95% confidence interval, the method has at least 95% specificity in identifying acute rejection (AR) relative to non-AR.

[0114] Some embodiments use a fixed threshold or a non-fixed threshold of dd-cfDNA per plasma volume, adjusted or scaled as described herein. The way to determine this threshold can be based on using a training data set to establish an algorithm to maximize performance. It can also consider other data, such as the patient's weight, age, or other clinical factors described herein.

[0115] In some embodiments, the method further includes using the amount of dd-cfDNA to determine the occurrence or possible occurrence of transplant rejection. In some embodiments, the amount of dd-cfDNA is compared with a cut-off threshold to determine the occurrence or possible occurrence of transplant rejection, wherein the cut-off threshold is adjusted or scaled according to the amount of total cfDNA. In some embodiments, the cut-off threshold is a function of the number of reads of dd-cfDNA.

[0116] In some embodiments, the method includes applying a scaled 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 labeling the sample if the amount of total cfDNA is higher than a predetermined value. In some embodiments, the method further includes labeling the sample if the amount of total cfDNA is lower than a predetermined value.

[0117] In some embodiments, the machine learning analysis further incorporates the time post-transplant as a parameter. In some embodiments, the machine learning analysis further incorporates the age of the transplant recipient and / or the transplant donor as a parameter. In some embodiments, the machine learning analysis further incorporates the pregnancy stage as a parameter. In some embodiments, the machine learning analysis further incorporates the number of fetuses as a parameter. In some embodiments, the machine learning analysis further incorporates the gender of the transplant recipient, the gender of the fetus, and / or the gender of the transplant donor as a parameter.

[0118] In some embodiments, the rejection risk 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 rejection risk 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 rejection risk of the transplant recipient is determined with 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.

[0119] Method for measuring the amount of nucleic acid

[0120] 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.

[0121] In some embodiments, the amount of dd-cfDNA is determined by using ratio-based and / or machine learning-artificial intelligence comparisons at single or multiple time points.

[0122] In some embodiments, the amount of cfDNA is measured by massively multiplex PCR (mmPCR) to obtain amplicons containing biomarkers, and the amplicons are sequenced.

[0123] In some embodiments, the amount of cfDNA is measured by using a microarray. In some embodiments, the amount of cfDNA is measured by using molecular barcoding and microscopy imaging (such as NanoString )

[0124] In some embodiments, the amount of dd-cfDNA is measured by: extracting cfDNA from a biological sample of a pregnant transplant recipient, wherein the extracted cfDNA includes dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification of the extracted DNA at 10 - 50,000 target loci in a single reaction volume, selected based on the homozygosity of the fetal parents; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of dd-cfDNA based on the sequencing reads.

[0125] In some embodiments, the method is performed without prior knowledge of the donor genotype. In some embodiments, the method does not include genotyping the transplant donor. In some embodiments, the paternal genotype of the fetus is known. In some embodiments, the method includes genotyping the biological father of the fetus.

[0126] In some embodiments, the amount of nucleic acid is measured by targeted amplification. In some embodiments, the amount of a specific cfDNA target is measured by targeted amplification. In some embodiments, targeted amplification includes PCR. In some embodiments, the primers for targeted amplification include 10 - 50,000, 100 - 50,000, 200 - 50,000, 500 - 20,000, or 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 pairs of forward and reverse PCR primers. In some embodiments, targeted amplification includes using 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 primer pairs to perform 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, 20,000 - 50,000 target loci in a single reaction volume to obtain an amplification product.

[0127] In some embodiments, targeted amplification includes nested PCR. In some embodiments, the primers for targeted amplification include a first universal primer and 10-50,000, 100-50,000, 200-50,000, 500-20,000, or 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-specific primers, and a second universal primer and 10-50,000, 100-50,000, 200-50,000, 500-20,000, or 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 internal target-specific primers. In some embodiments, targeted amplification includes using a first universal primer and 10-50,000, 100-50,000, 200-50,000, 500-20,000, or 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-specific primers to perform amplification at 10-50,000, 100-50,000, 200-50,000, 500-20,000, or 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 in a single reaction volume to obtain an amplification product.In some embodiments, targeted amplification includes using a second universal primer and 10 - 50,000, 100 - 50,000, 200 - 50,000, 500 - 20,000, or 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 internal target - specific primers to perform amplification at 10 - 50,000, 100 - 50,000, 200 - 50,000, 500 - 20,000, or 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 in a single reaction volume to obtain amplification products. In some embodiments, the methods disclosed herein include using at least 10, at least 100, at least 500, at least 1,000, at least 2,000, 10 - 1,000, 100 - 10,000, 200 - 50,000, 500 - 20,000 pairs of forward and reverse PCR primers to perform PCR amplification on at least 10, at least 100, at least 500, at least 1,000, at least 2,000 biomarkers, 10 - 1,000, 100 - 10,000, 200 - 50,000, or 500 - 20,000 RNA biomarkers. In some embodiments, step (b) includes using at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, at least 100 target RNA molecules, 2 - 10, 200 - 100, 50 - 500, or 50 - 2,000 pairs of forward and reverse PCR primers to amplify at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, at least 100 target RNA molecules, 2 - 10, 200 - 100, 50 - 500, or 50 - 2,000 target RNA molecules.

[0128] In some embodiments, the method further includes incorporating a tag into the amplification products before performing high - throughput sequencing, wherein the tag contains sequencing - compatible adapters. In some embodiments, the method further includes attaching a tag to the extracted DNA before performing targeted amplification, wherein the tag contains adapters for amplification. In some embodiments, the tag includes a sample - specific barcode, and wherein the method further includes pooling the amplification products from multiple samples before high - throughput sequencing and sequencing the amplification product pool together in a single run during the high - throughput sequencing.

[0129] In some embodiments, the amount of nucleic acid is determined by using, for example, a tracer nucleic acid or an internal calibration nucleic acid. The terms "tracer nucleic acid" or "internal calibration nucleic acid" are used interchangeably and refer to a composition of nucleic acids in which one or more of the following are pre-known: length, sequence, nucleotide composition, quantity, or biological origin. The tracer can be added to a biological sample derived from a subject to assist in estimating the amount of total cfDNA in the sample. It can also be added to a reaction mixture other than the biological sample itself.

[0130] For determining a cut-off threshold for transplant rejection

[0131] In some embodiments, the cut-off threshold is the estimated percentage of dd-cfDNA in total cfDNA or a function thereof. In some embodiments, the cut-off 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 cut-off threshold is adjusted according to the type of transplanted organ. In some embodiments, the cut-off threshold is adjusted according to the number of transplanted organs. In some embodiments, the cut-off threshold is adjusted according to the amount of fd-cfDNA in the sample.

[0132] In some embodiments, the cut-off threshold is proportional to the absolute dd-cfDNA concentration. In some embodiments, the cut-off threshold is the copy number of dd-cfDNA or a function thereof. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of dd-cfDNA. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of dd-cfDNA per unit volume of the blood sample. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of dd-cfDNA per unit volume of the blood sample multiplied by the body weight, BMI, or blood volume of the transplant recipient.

[0133] In some embodiments, the method further comprises longitudinally repeating the measurement of the amount of cfDNA and the amount of dd-cfDNA in the same transplant recipient, and determining the longitudinal change in the amount of cfDNA or a function thereof and the longitudinal change in the amount of dd-cfDNA or a function thereof.

[0134] Analysis of donor-derived cell-free DNA for monitoring transplant rejection

[0135] “Acute rejection or AR” is the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplanted tissue by immune cells of the recipient that carry out their effector functions and damage the transplanted tissue. The onset of acute rejection is rapid and typically occurs in humans within weeks after the transplant surgery. Usually, acute rejection can be inhibited or suppressed with immunosuppressive drugs such as rapamycin, cyclosporine A, anti-CD40L monoclonal antibody, etc.

[0136] “Chronic allograft injury or rejection” or “CAI” typically occurs in humans months to years after implantation, even when immunosuppression of acute rejection has been successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can generally be described by a specific set of conditions specific to a particular organ. For example, in lung transplantation, such conditions include fibroproliferative destruction of the airways (bronchiolitis obliterans); in heart transplantation or transplantation of heart tissue such as valve replacement, such conditions include fibrotic atherosclerosis; in kidney transplantation, such conditions include obstructive nephropathy, nephrosclerosis, and tubulointerstitial nephritis; and in liver transplantation, such conditions include vanishing bile duct syndrome. Chronic rejection is also characterized by ischemic injury, denervation of the transplanted tissue, hyperlipidemia, and hypertension associated with immunosuppressive drugs.

[0137] The term “transplant rejection” encompasses both acute and chronic transplant rejection. The term transplant rejection refers to a transplant in which the recipient and the donor are of the same species (“allograft rejection”) or of different species (“xenograft rejection”). The term “transplant injury” refers to all forms of graft dysfunction, regardless of the pathological diagnosis. The term “organ injury” refers to biomarkers that track organ dysfunction, whether the organ is native or transplanted and regardless of the cause.

[0138] In one 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 a biological sample of the transplant recipient, wherein the DNA comprises dd-cfDNA, fd-cfDNA, and rd-cfDNA; preparing a composition from the cfDNA, wherein one or more selected target loci are enriched, 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 the heterozygosity observed at the selected target loci in the extracted cfDNA is derived from the transplant; and quantifying the amount of the extracted cfDNA and the amount of dd-cfDNA based on the heterozygosity at the selected target loci to determine whether the amount of dd-cfDNA or a function thereof exceeds a cut-off threshold indicative of transplant rejection.

[0139] In one aspect, the present invention further includes: measuring the amount of dd-cfDNA in a biological sample obtained from a maternal transplant recipient, extracting DNA from the sample obtained from the transplant recipient, wherein the extracted DNA includes dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification of the extracted DNA at 10 - 50,000 target loci in a single reaction volume; sequencing the amplified DNA to obtain sequencing reads, and 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 cut-off threshold of the cfDNA amount indicating transplant rejection, wherein transplant rejection is determined based on whether the amount of dd-cfDNA or a function thereof exceeds the cut-off threshold indicating transplant rejection.

[0140] In another aspect, the present 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 includes dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification at 10 - 50,000 or 500 - 50,000 target loci in a single reaction volume using 10 - 50,000 or 500 - 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 not exceeding 100 bp; and quantifying the amount of dd-cfDNA in the amplification product.

[0141] In another aspect, the present 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 includes dd-cfDNA, fd-cfDNA, and rd-cfDNA; performing targeted amplification at 10 - 50,000 target loci in a single reaction volume using 10 - 50,000 primer pairs, wherein the target loci include polymorphic loci and non-polymorphic loci; sequencing the amplification product by high-throughput sequencing; and quantifying the amount of dd-cfDNA.

[0142] In a further aspect, the present invention relates to a method for determining the likelihood of transplant rejection in a recipient of a maternal transplant, 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 on the extracted cfDNA; performing targeted amplification at 10 - 50,000 target loci in a single reaction volume using 10 - 50,000 primer pairs, wherein the target loci comprise polymorphic loci and non-polymorphic loci; sequencing the amplification products by high-throughput sequencing; and quantifying the amount of dd-cfDNA in the blood sample, wherein a larger amount of dd-cfDNA indicates a greater likelihood of transplant rejection.

[0143] In some embodiments, the method further comprises performing universal amplification on the extracted DNA. In some embodiments, the universal amplification preferentially amplifies dd-cfDNA rather than rd-cfDNA and fd-cfDNA located from burst white blood cells.

[0144] 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, pig, primate, baboon, cow or dog.

[0145] In some embodiments, the transplant recipient has received a transplant selected from organ transplantation, tissue transplantation, cell transplantation and fluid transplantation. In some embodiments, the transplant recipient has received a transplant selected from the following: kidney transplantation, liver transplantation, pancreas transplantation, intestine transplantation, heart transplantation, lung transplantation, heart / lung transplantation, stomach transplantation, testis transplantation, penis transplantation, ovary transplantation, uterus transplantation, thymus transplantation, face transplantation, hand transplantation, leg transplantation, bone transplantation, bone marrow transplantation, corneal transplantation, skin transplantation, islet cell transplantation, heart valve transplantation, blood vessel transplantation and blood transfusion. In some embodiments, the transplant recipient has received a SPK transplant.

[0146] In some embodiments, the quantification step comprises determining the percentage of dd-cfDNA in the biological sample relative to the total of dd-cfDNA, fd-cfDNA and rd-cfDNA. In some embodiments, the quantification step comprises determining the copy number of dd-cfDNA per unit volume of the blood sample.

[0147] In some embodiments, the method further comprises using the quantified amount of dd-cfDNA to detect the occurrence or possible occurrence of active transplant rejection. In some embodiments, the method is performed without prior knowledge of the donor genotype.

[0148] In some embodiments, targeted amplification comprises simultaneously amplifying 10 to 50,000 target loci in a single reaction volume using (i) at least 10 to 50,000 different primer pairs, or (ii) at least 10 to 50,000 target-specific primers and 10 to 50,000 universal or tag-specific primers.

[0149] In some embodiments, each primer pair is designed to amplify a target sequence of about 50 bp to 100 bp. In some embodiments, each primer pair is designed to amplify a target sequence of no more than 75 bp. In some embodiments, each primer pair is designed to amplify a target sequence of about 60 bp to 75 bp. In some embodiments, each primer pair is designed to amplify a target sequence of about 65 bp.

[0150] In some embodiments, targeted amplification comprises amplifying at least 10 target loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 100 target loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 1,000 target loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 2,000 target loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 5,000 target loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 10,000 target loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying 10 to 10,000, 10 to 50,000, 100 to 50,000, or 1,000 to 50,000 target loci in a single reaction volume.

[0151] In some embodiments, the method further comprises measuring the amount of one or more alleles at a target locus that is a polymorphic locus. In some embodiments, polymorphic and non-polymorphic loci are amplified in a single reaction.

[0152] In some embodiments, the quantification step comprises detecting the amplified target loci using a microarray. In some embodiments, the quantification step does not comprise using a microarray.

[0153] In some embodiments, targeted amplification comprises simultaneously amplifying 10 to 50,000 target loci in a single reaction volume using (i) at least 10 to 50,000 different primer pairs, or (ii) at least 10 to 50,000 target-specific primers and 10 to 50,000 universal or tag-specific primers.

[0154] In a further aspect, the present invention relates to a method for diagnosing that a graft in a maternal transplant recipient is undergoing acute rejection, the method comprising: extracting DNA from a biological sample of the maternal transplant recipient, wherein the DNA comprises dd-cfDNA and rd-cfDNA; performing universal amplification on the extracted DNA; performing targeted amplification at 100 - 50,000 target loci in a single reaction volume using 100 - 50,000 primer pairs, wherein the target loci comprise polymorphic loci and non-polymorphic loci; sequencing the amplification products by high-throughput sequencing; 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 that the graft is undergoing acute rejection.

[0155] In some embodiments, the transplant rejection is antibody-mediated transplant rejection. In some embodiments, the transplant rejection is T cell-mediated transplant rejection.

[0156] 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 graft is undergoing borderline rejection, is undergoing other injury, or is stable.

[0157] In a further aspect, the present invention relates to a method for monitoring immunosuppressive therapy in a subject, the method comprising: extracting DNA from a biological sample of the transplant recipient, wherein the DNA comprises dd-cfDNA, rd-cfDNA and fd-cfDNA; performing universal amplification on the extracted DNA; performing targeted amplification at 10 - 50,000 target loci in a single reaction volume using 10 - 50,000 primer pairs, wherein the target loci comprise polymorphic loci 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 dd-cfDNA over a time interval indicates the graft status.

[0158] In some embodiments, the method further comprises adjusting the immunosuppressive therapy based on the dd-cfDNA level over a period of time.

[0159] In some embodiments, an increase in the dd-cfDNA level indicates transplant rejection and the need to adjust the immunosuppressive therapy. In some embodiments, no change or a decrease in the dd-cfDNA level indicates graft tolerance or stability and the need to adjust the immunosuppressive therapy.

[0160] In some embodiments, the method does not include genotyping the transplant donor and / or the transplant recipient.

[0161] In some embodiments, the target locus comprises 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.

[0162] In some embodiments, the extraction step comprises size selection to enrich dd-cfDNA and reduce the amount of rd-cfDNA seeded from bursting white blood cells.

[0163] In some embodiments, the universal amplification step preferentially amplifies dd-cfDNA rather than rd-cfDNA or fd-cfDNA.

[0164] In some embodiments, the method comprises longitudinally collecting multiple blood samples from the transplant recipient after transplantation and measuring the amounts of cfDNA and dd-cfDNA to determine the longitudinal changes in the amount of the transplant recipient's cfDNA or a function thereof and the longitudinal changes in the amount of dd-cfDNA. In some embodiments, the method comprises collecting and analyzing blood samples from the transplant recipient over a period of about three months, or about six months, or about twelve months, or about eighteen months, or about twenty-four months, etc. In some embodiments, the method comprises collecting blood samples from the transplant recipient at intervals of about one week, or about two weeks, or about three weeks, or about one month, or about two months, or about three months, etc.

[0165] In some embodiments, the method further comprises titrating the dose of the immunosuppressive therapy based on the total amount of cfDNA or the longitudinal changes in a function thereof and the amount of dd-cfDNA or the longitudinal changes in a function thereof.

[0166] In one aspect, the present disclosure relates to a method of administering an immunosuppressive therapy in a maternal transplant recipient, the method comprising: measuring the amount of cfDNA in a biological sample of the transplant recipient; measuring the amount of dd-cfDNA in a biological sample of the transplant recipient; and titrating the dose of the immunosuppressive therapy based on the amount of cfDNA or a function thereof and the amount of dd-cfDNA or a function thereof.

[0167] In some embodiments, the method has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sensitivity in identifying acute rejection (AR) compared to non-AR, wherein the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0168] In some embodiments, the method has 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% specificity in identifying AR compared to non-AR, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0169] 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 identifying AR compared to non-AR, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0170] In some embodiments, the method has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sensitivity in identifying AR compared to normal, stable allografts (STA), where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0171] In some embodiments, the method has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% specificity in identifying AR compared to STA, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0172] 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 identifying AR compared to STA, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0173] 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 is 10 times the LoD; the LoQ can be 5 times the LoD; the LoQ can be 1.5 times the LoD; the LoQ can be 1.2 times the LoD; the LoQ can be 1.1 times the LoD; or the LoQ can be equal to or greater than the LoD. In some embodiments, the LoB is equal to or less than 0.04%, the LoD is equal to or less than 0.05%, and / or the LoQ is equal to the LoD.

[0174] 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, where the linear value is an R 2 value, where R2 has a value of from about 0.98 to about 1.0. In some embodiments, R 2 has a value of 0.999. In some embodiments, the method has an accuracy determined by calculating a slope value and an intercept value by using linear regression of the measured donor fraction as a function of the corresponding attempted spike level, wherein 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 approximately 1, and the intercept value is approximately 0.

[0175] 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%.

[0176] In some embodiments, AR is antibody-mediated rejection (ABMR). In some embodiments, AR is T cell-mediated rejection (TCMR).

[0177] In some embodiments, there is provided a method of amplifying a target locus of dd-cfDNA from a biological sample of a maternal transplant recipient, the method comprising: extracting DNA from a blood sample of the transplant recipient, wherein the DNA comprises DNA derived from the transplanted cells, the transplant recipient, and the fetus; enriching the extracted DNA at the target locus, wherein the target locus comprises from 10 to 50,000 target loci comprising polymorphic loci and non-polymorphic loci; and amplifying the target locus.

[0178] In some embodiments, there is provided a method of detecting dd-cfDNA in a blood sample from a maternal transplant recipient, the method comprising: a) extracting DNA from a blood sample of the transplant recipient, wherein the DNA comprises cfDNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted DNA at the target locus, wherein the target locus comprises from 50 to 5000 target loci comprising polymorphic loci and non-polymorphic loci; c) amplifying the target locus; d) contacting the amplified target locus with a probe that specifically hybridizes to the target locus; and e) detecting the binding of the target locus to the probe, thereby detecting dd-cfDNA in the blood sample. In some embodiments, the probe is labeled with a detectable marker.

[0179] In some embodiments, a method for determining the likelihood of transplant rejection in a maternal transplant recipient is disclosed herein. The method includes: a) extracting DNA from a blood sample of the transplant recipient, wherein the DNA contains cfDNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted DNA at target loci, wherein the target loci contain from 50 to 5000 target loci comprising polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplanted DNA and the amount of transplant recipient DNA in the recipient's blood sample; wherein a larger amount of dd-cfDNA indicates a greater likelihood of transplant rejection.

[0180] In some embodiments, a method for monitoring immunosuppressive therapy in a subject is disclosed herein. The method includes: a) extracting DNA from a biological sample of the transplant recipient, wherein the DNA contains cfDNA derived from both the transplanted cells and the transplant recipient as well as the fetus; b) enriching the extracted DNA at target loci, wherein the target loci contain from 10 to 50,000 target loci comprising polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplanted DNA and the amount of recipient and fetal DNA in the recipient's biological sample; wherein a change in the dd-cfDNA level over a time interval indicates the transplant status. In some embodiments, the method further includes adjusting the immunosuppressive therapy based on the dd-cfDNA level over the time interval. In some embodiments, an increase in the dd-cfDNA level indicates transplant rejection and the need to adjust the immunosuppressive therapy. In some embodiments, a change or decrease in the dd-cfDNA level indicates transplant tolerance or stability and the need to adjust the immunosuppressive therapy.

[0181] In some embodiments, the methods disclosed herein require that the target loci be homozygous in the maternal transplant recipient and the biological father of the fetus, thereby ensuring homozygosity of the fetus at the target loci. Any heterozygosity detected in a biological sample from the maternal transplant recipient will be derived from the donor. The heterozygosity of the target loci can be used to quantify the amount of dd-cfDNA present in the biological sample.

[0182] Analysis method

[0183] In some embodiments, the method further includes obtaining genotype data from one or more of a transplant donor, a maternal transplant recipient, a fetus, and a biological father of the fetus. In some embodiments, obtaining genotype data from one or more of a transplant donor, a maternal transplant recipient, a fetus, and a biological father of the fetus includes preparing DNA from the donor, recipient, fetus, and father, wherein the preparation includes preferentially enriching the DNA at a plurality of polymorphic loci to obtain prepared DNA; optionally amplifying the prepared DNA; and measuring the DNA in the prepared sample at a 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.

[0184] In some embodiments, genetic data obtained from one or more of a transplant donor, a maternal transplant recipient, a fetus, and a biological father of the fetus is used to establish a joint distribution model of the expected allele count probabilities at a plurality of polymorphic loci on a chromosome. In some embodiments, a first sample has been isolated from the transplant recipient plasma, and wherein obtaining genotype data from the transplant recipient is accomplished by estimating the recipient genotype data based on the DNA measurements performed on the prepared sample.

[0185] In some embodiments, the preferential enrichment is such that the average degree of allele bias between the prepared sample and the first sample has a factor selected from the group consisting of: a factor of no more than 2, a factor of no more than 1.5, a factor of no more than 1.2, a factor of no more than 1.1, a factor of no more than 1.05, a factor of no more than 1.02, a factor of no more than 1.01, a factor of no more than 1.005, a factor of no more than 1.002, a factor of no more than 1.001, and a factor of no more than 1.0001. In some embodiments, the plurality of polymorphic loci are SNPs. In some embodiments, the DNA in the prepared sample is measured by sequencing.

[0186] In some embodiments, a diagnostic kit for assisting in determining the transplant status of a maternal transplant recipient is disclosed, wherein the diagnostic kit is capable of performing the preparation and measurement steps of the disclosed method.

[0187] In some embodiments, the allele count is probabilistic rather than binary. In some embodiments, measuring the DNA in the prepared sample at a plurality of polymorphic loci is intended to determine whether the graft has inherited one or more linked haplotypes.

[0188] In some embodiments, a joint distribution model of allele count probabilities is established by using data on the probability of crossovers at different positions of a chromosome within a chromosome to model the dependencies between polymorphic alleles on the chromosome. In some embodiments, the steps of establishing the joint distribution model of allele counts and determining the relative probabilities of each hypothesis are done using methods that do not require the use of a reference chromosome.

[0189] In some embodiments, determining the relative probability of each hypothesis utilizes the estimated fraction of dd-cfDNA in a prepared sample. In some embodiments, the DNA measurements from the prepared sample used to calculate allele count probabilities and determine the relative probabilities of each hypothesis include primary genetic data. In some embodiments, maximum likelihood estimation or maximum a posteriori estimation is used to perform the selection of the transplantation state corresponding to the hypothesis with the highest probability.

[0190] In some embodiments, determining the transplantation state further includes combining the relative probabilities of each state hypothesis in the state hypotheses determined using the joint distribution model and allele count probabilities with the relative probabilities of each state hypothesis in the state hypotheses calculated using statistical techniques selected from the group consisting of read count analysis, comparison of heterozygosity rates, statistical data available only when using donor genetic information, probabilities of normalized genotype signals for certain donor / recipient backgrounds, statistical data calculated using the estimated transplantation fraction of the first sample or the prepared sample, and combinations thereof.

[0191] In some embodiments, a confidence estimate of the determined transplantation state is calculated. In some embodiments, the method further includes taking a clinical measure based on the determined transplantation state.

[0192] In some embodiments, the method is used to generate a report showing the determined transplantation state. In some embodiments, a kit for determining the transplantation state is disclosed, the kit being designed to be used in conjunction with the methods disclosed herein, the kit including a plurality of internal forward primers and optionally a plurality of internal reverse primers and optionally additional chromosomes, wherein each of the primers is designed to hybridize to a DNA region directly upstream and / or downstream of one of the polymorphic loci on the target chromosome, wherein the hybridization region is separated from the polymorphic locus by a small number of bases, wherein the small number 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, 31 to 60, and combinations thereof.

[0193] In some embodiments, the cut-off threshold takes into account one or more of the following: donor genome copies / plasma volume, cfDNA yield / plasma volume, fetal genome copies / plasma volume, number of fetuses present, pregnancy stage, donor height, donor weight, donor age, donor sex, donor race, donor organ mass, donor organ, living donor vs. deceased donor, related donor vs. unrelated donor, recipient height, recipient weight, recipient age, recipient sex, recipient race, creatinine, eGFR (estimated glomerular filtration rate), cfDNA methylation, DSA (donor-specific antibody), KDPI (kidney donor profile index), medications (immunosuppressants, steroids, blood thinners, etc.), infections (BKV, EBV, CMV, UTI), recipient and / or donor HLA allele or epitope mismatches, Banff classification of kidney transplant pathology, and cause vs. surveillance or protocol biopsies.

[0194] In some embodiments, the cut-off threshold is scaled according to the amount of total cfDNA in the blood sample.

[0195] In some embodiments, when the dd-cfDNA amount is higher than the cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 80% sensitivity in identifying acute rejection (AR) relative to non-AR.

[0196] In some embodiments, when the dd-cfDNA amount is higher than the cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 70% specificity in identifying acute rejection (AR) relative to non-AR.

[0197] In some embodiments, when the dd-cfDNA amount is higher than the cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 80% sensitivity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the dd-cfDNA amount is higher than the cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 85% sensitivity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the dd-cfDNA amount is higher than the cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 90% sensitivity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the dd-cfDNA amount is higher than the cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 95% sensitivity in identifying acute rejection (AR) relative to non-AR.

[0198] In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 70% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 75% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 85% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 90% specificity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than a cut-off threshold scaled according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 95% specificity in identifying acute rejection (AR) relative to non-AR.

[0199] Multiplex amplification

[0200] In some embodiments, the method includes performing a multiplex amplification reaction prior to determining the sequences of the selectively enriched DNA to amplify multiple multi-target loci in one reaction mixture. In some embodiments, the target loci are selected at loci where both the maternal transplant recipient and the biological father of the fetus are homozygous.

[0201] In certain illustrative embodiments, nucleic acid sequence data is generated by high-throughput sequencing of multiple copies of a series of amplicons generated using a multiplex amplification reaction, where each amplicon of the series of amplicons spans at least one polymorphic locus in a set of polymorphic loci, and where each of the polymorphic loci in the set is amplified. For example, in these embodiments, multiplex PCR can be performed to amplify amplicons spanning 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 set up as a single reaction or as a pool of different subset multiplex reactions. The multiplex reaction methods provided herein, such as the large-scale multiplex PCR disclosed herein, provide exemplary procedures for performing the amplification reaction to help achieve improved multiplexing and thus sensitivity levels.

[0202] In some embodiments, amplification is performed using direct multiplex PCR, continuous PCR, nested PCR, double nested PCR, one-sided and semi-sided nested PCR, full nested PCR, one-sided full nested PCR, one-sided nested PCR, semi-nested PCR, semi-nested PCR, triple semi-nested PCR, semi-nested PCR, single-sided semi-nested PCR, reverse semi-nested PCR methods or single-sided PCR, which are described in U.S. Application No. 13 / 683,604, filed on November 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed on November 18, 2011, U.S. Publication No. 2012 / 0270212 and U.S. Serial No. 61 / 994,791, filed on May 16, 2014, all of which are hereby incorporated by reference in their entirety.

[0203] In some embodiments, multiplex PCR is used. In some embodiments, methods for amplifying target loci in a nucleic acid sample involve (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 produce a single reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to produce an amplification product that contains the target amplicons. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the targeted 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 amplification products are primer dimers. In some embodiments, the primers are in solution (such as dissolved in a liquid phase rather than a solid phase). In some embodiments, the primers are in solution and are not immobilized on a solid support. In some embodiments, the primers are not part of a microarray.

[0204] In certain embodiments, at least 1 / 2 of the multiplex amplification reactions are performed under limiting primer conditions. In some embodiments, limiting primer concentrations are used for 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2 or all of the multiplex reactions. Factors to consider when achieving limiting primer conditions in an amplification reaction (such as PCR) are provided herein.

[0205] In some embodiments, the multiplex amplification reaction can include, for example, a multiplex reaction between 2,500 and 50,000. In some embodiments, multiplex reactions are performed in the following ranges: the lower end of the range is between 10, 100, 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, 50,000, and the upper end of the range is between 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, 50,000, and 100,000.

[0206] In one embodiment, the multiplex PCR assay is designed to amplify potential heterozygous SNPs 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 between 10 and 200 PCR assays, between 200 and 1,000 PCR assays, between 1,000 and 5,000 PCR assays, or between 5,000 and 20,000 PCR assays (10-plex to 200-plex, 200-plex to 1,000-plex, 1,000-plex to 5,000-plex, 5,000-plex to 20,000-plex, greater than 20,000-plex, respectively). In one embodiment, a multiplex pool of at least 10,000 PCR assays (10,000-plex) is designed for a single reaction to amplify potential heterozygous SNP loci to amplify cfDNA obtained from blood, plasma, serum, solid tissue, or urine samples. The SNP frequency of each locus can be determined by sequencing the amplicons by cloning or some other method. In another embodiment, the original cfDNA sample is divided into two samples, and a parallel 5,000-plex assay is performed. In another embodiment, the original cfDNA sample is divided into n samples, and a parallel (about 10,000 / n)-plex assay is performed, where n is between 2 and 12, or between 12 and 24, or between 24 and 48, or between 48 and 96.

[0207] In one embodiment, the methods disclosed herein use efficient, highly multiplexed targeted PCR amplification of DNA followed by high-throughput sequencing to determine allele frequencies at each target locus. A technique that allows highly multiplexed targeted PCR to be performed in an efficient manner involves designing primers that are unlikely to hybridize to each other. PCR probes, commonly referred to as primers, are selected by creating a thermodynamic model 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 potential primer pairs or unexpected interactions between the primers and the sample DNA, and then using the model to eliminate designs that are incompatible with other designs in the pool. Another technique that allows highly multiplexed targeted PCR to be performed in an efficient manner is to use a partially or fully nested approach for targeted PCR. Using one or a combination of these methods allows 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, and the resulting amplified DNA includes most of the DNA molecules that will map to the target locus upon sequencing. Using one or a combination of these methods allows multiplexing of a large number of primers in a single pool, and the resulting amplified DNA includes greater than 50%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99% of the DNA molecules that map to the target locus.

[0208] Genetic data obtained from multiplex PCR is analyzed using bioinformatics methods. 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.

[0209] High-throughput sequencing

[0210] In some embodiments, the sequence of the amplicons is determined by performing high-throughput sequencing.

[0211] Genetic data of a transplant recipient and / or a transplant donor can be converted from a molecular state to an electronic state by measuring appropriate genetic material using tools and / or techniques from the following group, which includes but is not limited to: genotyping microarrays and high-throughput sequencing. Some high-throughput sequencing methods include Sanger DNA sequencing, pyrosequencing, the ILLUMINA SOLEXA platform, the ILLUMINA GENOME ANALYZER or the 454 sequencing platform of APPLIED BIOSYSTEM, the TRUE SINGLE MOLECULE SEQUENCING platform of HELICOS, the electron microscopy sequencing method of HALCYON MOLECULAR, PacBio, Oxford Nanopore, or any other sequencing method. In some embodiments, high-throughput sequencing is performed on Illumina All such methods physically transform the genetic data stored in a DNA sample into a set of genetic data, which is typically stored in a memory device for processing.

[0212] In some embodiments, the sequence of selectively enriched DNA is determined by performing microarray analysis. In one embodiment, the microarray can be an ILLUMINA SNP microarray or an AFFYMETRIX SNP microarray.

[0213] In some embodiments, the sequence of selectively enriched DNA is determined by performing quantitative PCR (qPCR) or digital droplet PCR (ddPCR) analysis. qPCR measures the fluorescence intensity at specific times (generally after each amplification cycle) to determine the relative quantity of the target molecule (DNA). ddPCR measures the actual quantity of the molecule (target DNA) since each molecule is in a droplet, making it a discrete "digital" measurement. It provides absolute quantification because ddPCR measures the positive fraction of the sample, i.e., the number of droplets that fluoresce due to correct amplification. This positive fraction accurately indicates the initial amount of the template nucleic acid.

[0214] Definitions

[0215] As used herein, the term "single nucleotide polymorphism (SNP)" refers to a single nucleotide at which there may be a difference between the genomes of two members of the same species. The use of the term does not imply any limitation on the frequency of occurrence of each variant.

[0216] In some embodiments, for example, a sequence refers to a DNA sequence, an RNA sequence, or a genetic sequence. A sequence can refer to the basic physical structure of a DNA or RNA molecule or strand in an individual. A sequence can refer to the nucleotide sequence found in a DNA or RNA molecule, or the complementary strand of a DNA or RNA molecule. A sequence can refer to the information contained in a DNA or RNA molecule, as its bioinformatics representation.

[0217] In some embodiments, for example, a locus refers to a specific region of interest on an individual's DNA or RNA, including but not limited to one or more SNPs, sites where insertions or deletions may occur, or sites of some other relevant genetic variations. A disease-related SNP can also refer to a disease-related locus.

[0218] In some embodiments, for example, a polymorphic allele, also referred to as a "polymorphic locus", refers to an allele or locus where the genotype varies among individuals of a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms (SNPs), short tandem repeats, deletions, duplications, and inversions.

[0219] In some embodiments, for example, an allele refers to a nucleotide or nucleotide sequence that occupies a specific locus.

[0220] In some embodiments, for example, genetic data, also referred to as "genotype data", refers to data that describes aspects of the genomes of one or more individuals. It can refer to one or a set of loci, a partial or entire sequence, a partial or entire chromosome, or an entire genome. It can refer to the identity of one or more nucleotides; it can refer to a collection of consecutive nucleotides, or nucleotides from different positions in the genome, or a combination thereof. Genotype data is typically bioinformatics, however, the physical nucleotides in a sequence can also be considered chemically encoded genetic data. Genotype data can be said to be "on", "of", "at", "from", or "on" an individual. Genotype data can refer to output measurements from a genotyping platform that are made on genetic material.

[0221] In some embodiments, for example, genetic material, also referred to as a "genetic sample", refers to the physical material from one or more individuals that includes nucleic acids (e.g., including DNA or RNA), such as tissue or blood.

[0222] In some embodiments, for example, allele data refers to a set of genotype data regarding a set of one or more alleles. It can refer to phased, haplotype data. It can refer to SNP identity, and can also refer to sequence data of nucleic acids, including insertions, deletions, duplications, and mutations.

[0223] In some embodiments, an allelic state refers to the actual state of a gene in a set of one or more alleles. It can refer to the actual state of a gene as described by allelic data.

[0224] In some embodiments, an allelic ratio or allele ratio refers to the ratio between the amounts of each allele at a locus present in a sample or an individual. When a sample is measured by sequencing, the allelic ratio can refer to the ratio of sequence reads mapped to each allele at the locus. When a sample is measured by an intensity-based measurement method, the allelic ratio can refer to the ratio of the amounts of each allele present at the locus estimated by the measurement method.

[0225] In some embodiments, for example, an allelic count refers to the number of sequences mapped to a specific locus, and if the locus is polymorphic, it refers to the number of sequences mapped to each allele among the alleles. If each allele is counted in a binary manner, the allelic count will be an integer. If the alleles are counted probabilistically, the allelic count can be a fraction.

[0226] In some embodiments, for example, a primer, also referred to as a "PCR probe", refers to a single DNA molecule (DNA oligomer) or a collection of DNA molecules (DNA oligomers), where the DNA molecules are the same or approximately the same, and where the primer contains a region designed to hybridize to a targeted polymorphic locus and contains a primer sequence designed to allow amplification such as PCR amplification. The primer can also contain a molecular barcode. The primer can contain a random region that is different for each individual molecule.

[0227] In some embodiments, for example, a hybridization capture probe refers to any nucleic acid sequence (possibly modified) generated by various methods such as PCR or direct synthesis, and is designed to be complementary to one strand of a specific target DNA or RNA sequence in a sample. Exogenous hybridization capture probes can be added to a prepared sample and hybridized through a denaturation and reannealing process to form duplexes of exogenous and endogenous fragments. Then, these duplexes can be physically separated from the sample in various ways.

[0228] In some embodiments, for example, a sequence read refers to data representing a nucleotide base sequence that is measured using a clone sequencing method. Clone sequencing can generate sequence data representing a single original DNA or RNA molecule, or its clone or cluster. A sequence read can also have an associated quality score at each base position of the sequence, indicating the probability that the nucleotide is correctly determined.

[0229] In some embodiments, for example, mapping sequence reads is the process of determining the source location of a sequence read in a genomic sequence of a particular organism. The source location of the sequence read is based on the nucleotide sequence similarity of the read to the genomic sequence.

[0230] In some embodiments, for example, donor-derived DNA or RNA refers to DNA or RNA that was originally part of a cell that has a genotype substantially identical to that of the transplant donor. The donor can be a human or non-human mammal (e.g., a pig).

[0231] In some embodiments, for example, recipient-derived DNA or RNA refers to DNA or RNA that was originally part of a cell that has a genotype substantially identical to that of the transplant recipient.

[0232] In some embodiments, DNA can 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.

[0233] In some embodiments, RNA can 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 miscellaneous RNA (miscRNA). In some embodiments, the RNA is cell-free RNA. In some embodiments, the cell-free RNA is derived from exosomes or microvesicles.

[0234] In some embodiments, the amplification of RNA includes reverse transcription of the RNA to produce complementary DNA (cDNA), followed by amplification of the cDNA by amplification methods disclosed elsewhere herein.

[0235] In some embodiments, for example, transplant recipient plasma refers to the plasma portion of the blood from a female patient (e.g., an organ transplant recipient) who has received an allograft or xenograft.

[0236] As used herein, the terms "maternal transplant recipient", "pregnant 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, e.g., an organ transplant recipient. In some embodiments, the maternal transplant recipient may no longer be pregnant, but fetal-derived nucleic acids (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 fetus of the maternal transplant recipient. In some embodiments, paternal DNA refers to DNA from the biological father of the fetus.

[0237] In some embodiments, the biological sample can be blood, plasma, saliva, semen, sperm, cell culture supernatant, mucus secretion, dental plaque, gastrointestinal tissue, feces, urine, hair, bone, body fluid, tear, tissue, skin, nail, blastomere, embryo, amniotic fluid, chorionic villus sample, bile, lymph, cervical mucus, or forensic sample.

[0238] 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 percentage of DNA or RNA molecules corresponding to the locus in the DNA or RNA mixture after enrichment than in the DNA or RNA mixture before enrichment. The technique can involve selective amplification of DNA or RNA molecules corresponding to the locus. The technique can involve removal of DNA or RNA molecules that do not correspond to the locus. The technique can involve a combination of methods. The enrichment level is defined as the percentage of DNA or RNA molecules corresponding to the locus in the mixture after enrichment divided by the percentage of DNA or RNA molecules corresponding to the locus in the mixture before enrichment. Preferential enrichment can be performed at multiple loci. In some embodiments of the present disclosure, the enrichment level is greater than 20. In some embodiments of the present disclosure, the enrichment level is greater than 200. In some embodiments of the present disclosure, the enrichment level is greater than 2,000. When preferential enrichment is performed at multiple loci, the enrichment level can refer to the average enrichment level of all loci in the set of loci.

[0239] In some embodiments, for example, amplification refers to a technique for increasing the copy number of DNA and / or RNA molecules.

[0240] In some embodiments, for example, selective amplification can refer to a technique for increasing the copy number of specific DNA and / or RNA molecules or DNA and / or RNA molecules corresponding to specific DNA and / or RNA regions. Selective amplification can also refer to a technique for increasing the copy number of specific targeted DNA and / or RNA molecules or targeted DNA and / or RNA regions more than the copy number of non-targeted DNA and / or RNA molecules or regions. Selective amplification can be a method of preferential enrichment.

[0241] In some embodiments, for example, a universal primer sequence refers to a DNA sequence that can be appended to a population of target nucleic acid molecules, such as by ligation, PCR, or ligation-mediated PCR. Once added to the target molecule population, a universal primer sequence-specific primer can be used to amplify the target population using a single pair of amplification primers. The universal primer sequence does not need to be related to the target sequence.

[0242] In some embodiments, for example, a universal adapter or "ligation adapter" or "library tag" is a DNA molecule containing a universal primer sequence that can be covalently linked to the 5'-major end and 3'-major end of a population of target double-stranded DNA molecules. The addition of the adapter provides universal primer sequences at the 5'-major end and 3'-major end of the target population from which PCR amplification can be performed to amplify all molecules of the target population using a single pair of amplification primers.

[0243] In some embodiments, for example, targeting refers to a method for selectively amplifying or otherwise preferentially enriching those DNA or RNA molecules corresponding to a set of loci in a DNA or RNA mixture.

[0244] Working Examples

[0245] Example 1

[0246] This example is illustrative only, and those skilled in the art will understand that the invention disclosed herein can be practiced in a variety of other ways.

[0247] Blood samples

[0248] Adult pregnant or young adult patients receive donor organs from related or unrelated living donors or unrelated deceased donors. The time points for blood sampling from the patients after transplantation surgery are at the time of transplant biopsy or at various pre-specified time intervals based on laboratory protocols. Optionally, the samples are biopsy-matched and blood is drawn at the time of clinical dysfunction and biopsy or at protocol biopsy (when most patients do not have clinical dysfunction). Additionally, some patients have serial blood draws after transplantation.

[0249] Nucleic acid measurement in blood samples

[0250] 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, especially cfDNA, are extracted from plasma samples using the QIAamp TM Circulating Nucleic Acid Kit (Qiagen), and quantified using the LabChip TM NGS 5k Kit (Perkin Elmer, Waltham, MA, USA). 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, and 18 cycles of library amplification are modified to stabilize the library. The purified library is quantified using LabChip TM NGS 5k, as described in Abbosh et al., Nature 545:446-451 (2017). Target enrichment is accomplished using large-scale multiplex PCR (mmPCR), which uses a modified version of Zimmermann et al., Prenat. Diagn. 32:1233-1241 (2012), which is incorporated herein by reference in its entirety, and 13,392 single nucleotide polymorphisms (SNPs) are targeted. Then, the amplicons are sequenced on an Illumina HiSeq 2500 Rapid for 50 cycles single-end, with 10 to 11 million reads per sample.

[0251] Example 2

[0252] Determining the transplant status of maternal transplant recipients

[0253] Dd-cfDNA is measured in 6 pregnant kidney recipients, as Figure 1 shown. Identifying SNPs that are homozygous in both the mother and father ensures fetal homozygosity, and the heterozygosity observed in maternal cfDNA is derived from the transplant. These SNPs are used to calculate the donor fraction estimate (DFE). The clinical data collected include kidney function tests, mode of delivery, preeclampsia, and prior hypertension.

[0254] The DFE is calculated for 4 / 6 pregnant kidney transplant recipients. Among these patients, 5 / 6 had prior chronic hypertension, and 4 / 6 had superimposed preeclampsia. All patients underwent medically indicated induction of labor, followed by cesarean section for various indications. Medical record review also revealed that during the study period, serum creatinine and acute kidney injury spiked in all patients before, after, or during delivery.

[0255] This study demonstrated that cell-free DNA of donor origin (dd-cfDNA) can be measured in pregnant kidney transplant recipients with chronic kidney dysfunction. These measurements can help to distinguish between acute rejection and hypertensive disorders of pregnancy. A larger cohort study will confirm the utility of longitudinal monitoring during pregnancy to inform clinicians in clinical decisions that may impact transplant outcomes and maternal-fetal health.

[0256] Example 3: Cell-free DNA of donor origin (dd-cfDNA) in pregnant kidney transplant recipients

[0257] Background. In pregnant kidney transplant recipients (KTRs), the symptoms of early preeclampsia and kidney transplant rejection cannot be distinguished non-invasively. Cell-free DNA of donor origin (dd-cfDNA) is a biomarker that can be measured non-invasively to assess the risk of allograft rejection. The Prospera TM test uses a single nucleotide polymorphism (SNP)-based massively multiplex PCR (mmPCR) method to measure dd-cfDNA in KTRs. In adult non-pregnant KTRs, detection of dd-cfDNA ≥ 1% is associated with rejection. Measuring dd-cfDNA in pregnant KTRs requires distinguishing donor cfDNA fragments from fetal and maternal cfDNA fragments. The present inventors have successfully demonstrated the measurement of dd-cfDNA in a small cohort of pregnant KTRs with hypertensive disorders and describe here two cases with medical details.

[0258] Methods. Six pregnant KTRs were included in this study. Genomic DNA (gDNA) from the father and total cfDNA from the mother were extracted from oral swabs and blood draws, respectively (Figure 2A). The dd-cfDNA fraction was measured in pregnant KTRs using an integrated (Panorama TM / Prospera TM ) bioinformatics and laboratory workflow that combines sequencing of total maternal cfDNA and paternal gDNA ( Figure 2B ). Clinical data were collected, including kidney function tests, mode of delivery, serum creatinine, preeclampsia, prior hypertension, and clinical history.

[0259] Results. dd-cfDNA was calculated in 4 / 6 pregnant KTRs; 5 / 6 patients had a history of chronic hypertension, and 4 / 6 had superimposed preeclampsia. All patients underwent medically indicated labor induction, followed by cesarean section for various indications. During the study period, serum creatinine (SCr) and acute kidney injury (AKI) spiked in all patients before, after, or during delivery. Patient 1 (Figure 3A) was diagnosed with AKI due to pre-pregnancy tacrolimus toxicity and underwent labor induction at gestational age 38w5d due to hypertension and elevated SCr levels. Three months prior to AKI diagnosis, the patient's dd-cfDNA was 1.16%. Throughout pregnancy and the postpartum period, patient 2( Figure 3B ) had a sharp increase in SCr levels. During pregnancy, the patient was suspected of having acute KT rejection due to hypertension, and dd-cfDNA was 0.27% at around gestational age 15w. Complications associated with possible KT rejection prompted labor induction at gestational age 27w1d. Postpartum, the patient was diagnosed with AKI with preeclampsia and malignant hypertension due to non-compliance.

[0260] Conclusions. This proof-of-concept study demonstrates that dd-cfDNA can be measured in pregnant KTRs with chronic kidney dysfunction. These measurements help distinguish between acute rejection and hypertensive disorders of pregnancy. Further validation of dd-cfDNA testing in pregnant KTRs will help provide information on allograft and maternal-fetal health to physicians.

[0261] Example 4: Donor-derived cell-free DNA (dd-cfDNA) in pregnant kidney recipients

[0262] Successful kidney transplantation restores fertility and enables women of childbearing age who are kidney recipients to become pregnant successfully. Pregnant women who have received kidney transplants are at higher risk of maternal-fetal complications, including miscarriage, preterm birth, hypertensive disorders of pregnancy, and graft function deterioration and rejection, and the rate of kidney transplantation in this population continues to increase. Graft function is crucial for both the pregnant woman and the neonate, as well as for the life course of the allograft. During pregnancy, the ability to distinguish between preeclampsia and kidney transplant rejection is crucial because the management, timing of birth, and implications of the diagnosis are very different.

[0263] A definitive diagnostic test for kidney rejection requires a kidney biopsy, which is only recommended if the histological diagnosis would alter the management during pregnancy. Traditionally, the monitoring of kidney allografts has involved assessing serum creatinine levels, as well as monitoring blood pressure and evaluating proteinuria. These parameters are used as indirect indicators of changes in allograft function and can suggest the possibility of acute rejection. However, these parameters are relatively non-specific markers and can be affected by factors other than rejection, such as dehydration, gestational age, and preeclampsia. More recently, donor-derived cell-free DNA (dd-cfDNA) has emerged as a non-invasive and specific biomarker for allograft rejection.

[0264] Currently, it is not feasible to non-invasively distinguish allograft rejection, kidney disease progression, and preeclampsia in pregnant kidney transplant recipients without performing a kidney biopsy. Monitoring the allograft status in pregnant kidney transplant recipients (KTRs) using dd-cfDNA requires the ability to distinguish fetal / placental, donor, and recipient cfDNA fragments. This study aimed to measure donor-derived cfDNA in a cohort of pregnant kidney transplant recipients using a combination of placental-derived cfDNA and a transplantation monitoring technique that uses dd-cfDNA in combination with paternal DNA swabs. The study here demonstrated the feasibility of using dd-cfDNA during pregnancy in a cohort of pregnant women with hypertensive disorders.

[0265] Methods. This study included blood samples from pregnant women who received kidney transplants during pregnancy. Pregnant patients who received kidney allografts were tested with Prospera TM (Natera, Inc.) (n = 9). Prospera TM is a transplantation rejection detection and monitoring test that detects cell-free DNA (dd-cfDNA) from the allograft donor in the blood. The dd-cfDNA is expressed as a fraction of the total cell-free DNA (cfDNA). This clinical test is a large-scale 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 dd-cfDNA fraction of ≥1% cutoff to indicate active rejection. However, the calculation of the donor fraction estimate in pregnant women is more complex because the cfDNA in the mother's peripheral blood is a mixture of the mother's cfDNA, donor-derived cfDNA from the transplanted organ, and fetal cfDNA from maternal and paternal sources. To overcome this problem, first, with the modified Prospera TMThe workflow takes genomic DNA (gDNA) as input and sequences oral swabs from the father to identify the paternal genotype. Genomic DNA (n = 6) is isolated using the Qiagen Dneasy Blood and Tissue Kit and quantified by Qubit dsDNA HS (Thermo). The gDNA is sheared to approximately 160 bp using a Covaris LE220 sonicator. The sheared samples are quantified, normalized, and library preparation is performed. After mmPCR, the barcoded samples are pooled, quantified, and size fractionated on a 2100 Bioanalyzer (Agilent) and sequenced on a NextSeq 500. The sequencing data is processed through a custom Prospera TM bioinformatics pipeline.

[0266] Donor-derived cfDNA is determined from the transplanted kidney. The cfDNA of the patient is sequenced starting from peripheral blood samples using the Prospera TM laboratory workflow, and donor-derived cell-free DNA (dd-cfDNA) is measured in 6 pregnant kidney recipients (Figure 2). Custom bioinformatics analysis is performed to quantify the donor-derived cfDNA (Figure 4). To determine the maternal genotype, any homozygous SNP where more than 75% of the reads belong to one allele is considered homozygous for the mother. To determine the paternal genotype, any homozygous SNP from the paternal oral swab where more than 99% of the reads belong to one allele is considered homozygous. If the maternal and paternal genotypes are homozygous for the same allele, then the fetal genotype must also be. After filtering out SNPs where the mother and fetus have all four copies of the same allele, the filtered set of SNPs is run through the Prospera TM algorithm to determine the dd-cfDNA percentage (Figure 4). The clinical data collected includes kidney function tests, mode of delivery, preeclampsia, and previous hypertension.

[0267] Results. Nine pregnant women were recruited. Among the nine participants with known and confirmed paternity, six received and obtained paternal oral swabs. These six participants were included in the study. Table 1 presents the details. Figure 4 presents two representative adjudicated cases. Donor-derived cfDNA analysis was performed using Prospera with a custom data analysis protocol, resulting in 4 patients at risk of rejection (Figure 5). In two cases, it may not be possible to distinguish the donor cfDNA because the donor is the father of the fetus or the twin sister of the recipient. In 3 of the remaining 4 cases, the DFE was significantly lower than 1% and was therefore reported as low risk, but in 2 cases it was above the 1% threshold. Case 2 also had an abnormally high total cfDNA amount.

[0268] Discussion. Pregnancy in women undergoing dialysis is rare, with a low incidence of conception ranging from 0.9% to 7%, and a high incidence of preterm birth. Due to deteriorating renal function, the fertility of these women may be impaired, and kidney transplantation has a restorative effect on ovulation and fertility. However, although most pregnancies after kidney transplantation result in live births, the risk of fetal complications is high, including preterm birth, low birth weight, and fetal growth restriction (FGR). The evidence regarding the live birth rate in pregnant kidney transplant recipients is conflicting, with some studies showing a live birth rate comparable to that of the general population, while others report a lower live birth rate. During pregnancy, it is crucial to maintain adequate immunosuppression and adjust it according to pregnancy-related pharmacokinetic and physiological changes.

[0269] For these reasons, it is recommended that pregnant kidney transplant recipients be closely monitored by both a transplant nephrologist and a high-risk obstetrician during pregnancy. Monitoring should cover the monitoring of hypertension, preeclampsia, gestational diabetes, kidney allograft dysfunction, and infection, with the main goal being to achieve a near-term or full-term pregnancy without hypertensive complications, allograft dysfunction, or rejection.

[0270] In recent years, the use of cell-free DNA (cfDNA) in prenatal care has generated great interest. In the context of pregnancy, cfDNA (also known as non-invasive prenatal testing) can detect fetal aneuploidies (especially trisomies 21, 18, and 13 and sex chromosomes). Additionally, monitoring donor-derived cfDNA (dd-cfDNA) has been shown to be a promising non-invasive method for detecting early signs of graft rejection in transplant recipients. Studies indicate that when dd-cfDNA levels exceed 1%, the likelihood of acute kidney rejection increases.

[0271] Given these observations, it is reasonable to hypothesize that there is a correlation between the fraction of dd-cfDNA and the occurrence of rejection in pregnant kidney recipients, and that by continuously monitoring dd-cfDNA levels, individuals at higher risk of these complications can be identified, enabling closer monitoring and timely intervention, and potentially serving as a useful tool for differentiating acute rejection from preeclampsia without the need for a kidney biopsy. Using dd-cfDNA and paternal oral DNA swabs, and transplant monitoring techniques, this study successfully measured dd-cfDNA in a cohort of pregnant kidney transplant recipients. In 2 cases, the donor was the mother's 2-haplotype sister or the father of the child. In the remaining 4 cases, 3 cases were determined to be at low rejection risk, and 1 case was reported as possibly high risk (DFE > 1%).

[0272] In summary, this study demonstrated that dd-cfDNA can be measured in pregnant kidney recipients and that kidney rejection can be distinguished from hypertensive disorders of pregnancy in a specific high-risk kidney transplant recipient population. This non-invasive alternative blood marker can help distinguish acute rejection from hypertensive disorders of pregnancy. The presence of cfDNA in pregnant women has potentially been shown to be a diagnostic and monitoring tool for transplant rejection. Exploring the correlation between the fraction of cfDNA and the occurrence of rejection in pregnant kidney transplant recipients holds promise for improving risk assessment and patient management. This study may have a significant impact on nearly 3,500 women of childbearing age in the United States who receive solid organ transplants.

[0273] ****

Claims

1. A method for preparing an amplified DNA composition, wherein the DNA is derived from a biological sample of a maternal transplant recipient, the method being for determining the transplant status, the method comprising: (a) extracting cell-free DNA (cfDNA) from the biological sample of the maternal transplant recipient, wherein the extracted cfDNA comprises 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 a fetus; (b) preparing a composition from the cfDNA, wherein a plurality of target loci are enriched, wherein the target loci comprise one or more SNP loci at which the maternal transplant recipient and the biological father of the fetus are homozygous to ensure homozygosity of the fetus at the SNP loci, such that the heterozygosity observed at the SNP loci in the extracted cfDNA is derived from the transplant; and (c) quantifying the amount of dd-cfDNA based on the heterozygosity at the SNP loci to determine whether the amount of dd-cfDNA or a function thereof exceeds a cut-off threshold indicative of transplant rejection.

2. The method according to claim 1, wherein quantifying the cfDNA and dd-cfDNA comprises 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 or claim 2, wherein the target loci comprise 10 - 50,000 target loci, and the method further comprises performing multiplex targeted amplification of the DNA at the 10 - 50,000 target loci in a single reaction volume.

4. The method according to claims 1 to 3, wherein step (c) further comprises determining the amount of the allele of transplant origin at the one or more SNP loci, and determining whether the amount of the allele of transplant origin at the one or more SNP loci or a function thereof exceeds a cut-off threshold indicative of transplant rejection; wherein transplant rejection is determined by a combination of (i) the amount of the allele of transplant origin at the one or more SNP loci or a function thereof and (ii) the total amount of dd-cfDNA or a fraction of dd-cfDNA.

5. A method for administering an 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 of the transplant recipient according to the method of any one of claims 1 to 5; and (b) titrating the dose of the immunosuppressive therapy according to the amount of cfDNA or a function thereof and the amount of dd-cfDNA or a function thereof.

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

7. The method according to claim 6, further comprising titrating the dose of the immunosuppressive therapy based on 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.

8. The method according to any one of claims 5 to 7, wherein: an increase in the dd-cfDNA level indicates transplant rejection and the need to adjust the immunosuppressive therapy; and a change or decrease in the dd-cfDNA level indicates transplant tolerance or stability and the need to adjust the immunosuppressive therapy.

9. The method according to any one of claims 1 to 8, further comprising sequencing the paternal DNA of the biological father of the fetus and identifying one or more SNP loci at which the maternal transplant recipient and the biological father of the fetus are homozygous while the dd-cfDNA contains heterozygous alleles.

10. The method according to any one of claims 1 to 9, wherein the method is performed without prior knowledge of the donor and / or recipient genotypes.

11. The method according to any one of claims 1 to 10, further comprising universal amplification of the extracted DNA.

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

13. The method according to any one of claims 1 to 12, wherein the extraction step includes size selection to enrich dd-cfDNA and reduce the amounts of rd-cfDNA and fd-cfDNA.

14. The method according to any one of claims 1 to 13, wherein the amount of cfDNA is measured by quantitative PCR, real-time PCR, digital PCR, sequencing, microarray, or molecular barcoding and microscopy imaging.

15. The method according to any one of claims 1 to 14, wherein the amount of dd-cfDNA is determined by using ratio-based and / or machine learning-artificial intelligence comparisons at single or multiple time points.

16. The method according to any one of claims 1 to 15, wherein the cut-off threshold is the estimated percentage of dd-cfDNA in total cfDNA or a function thereof.

17. The method according to any one of claims 1 to 16, wherein an amount of dd-cfDNA greater than 1% of the total cfDNA indicates that the transplant is undergoing acute rejection, and wherein an amount of dd-cfDNA less than 1% of the total cfDNA indicates that the transplant is undergoing critical rejection, is undergoing other injury, or is stable.

18. The method according to any one of claims 1 to 17, 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, islet cells, heart valve, blood vessel, and blood transfusion.

19. The method according to any one of claims 1 to 18, wherein the sample is obtained from the transplant recipient less than 18 months after transplantation.

20. The method according to any one of claims 1 to 19, wherein logistic regression, random forest or decision tree machine learning analysis is used to determine the rejection risk of the transplant recipient.

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

22. The method according to any one of claims 1 to 21, wherein the biological sample is blood, serum, plasma or urine.

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