Method for determining and monitoring xenograft rejection by measuring nucleic acids or proteins derived from xenograft

Abstract

The present disclosure provides a method for the preparation and analysis of a biological sample of a xenograft receptor wherein the method comprises extracting fragmented or intact cell-free DNA, RNA (such as mRNA or miRNA) or protein of a sample derived from the xenograft receptor, and measuring the amount of cell-free DNA, RNA (such as mRNA or miRNA) or protein derived from said xenograft enables the assessment of xenograft rejection. The detection of the cell-free DNA or RNA can be carried out by preparing a sequencing library and carrying out whole genome sequencing.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 413,738, filed on October 6, 2022, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Rapid detection of graft injury and / or rejection remains a challenge for transplant recipients. Xenotransplantation, which involves transplanting animal organs into human patients with end-stage organ failure, is a potential approach to alleviate the chronic shortage of transplantable organs. Several efforts are underway to introduce this approach into clinical practice, including generating transgenic donor animals with reduced immunogenicity and a lower risk of transmitting animal viruses to humans, as well as conducting clinical trials of transplanting animal organs into human patients. The rapid progress of this research and the severe shortage of human organs available for transplantation make it possible for xenotransplantation to become a major approach for treating end-stage organ failure in the clinic in the near future.

[0004] After xenotransplantation, 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. Traditional biopsy-based tests are invasive and costly, and may lead to late diagnosis of graft injury and / or rejection. Therefore, there is a need for a non-invasive test for xenograft injury and / or rejection that enables early diagnosis of graft injury and / or rejection and is more sensitive and specific than traditional biopsy-based tests.

[0005] Accordingly, there is a need for improved methods for accurately diagnosing, screening, testing, and monitoring xenograft injury and / or rejection at an early stage. The present disclosure addresses this need. SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure relates to a method for preparing a DNA composition derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient for determining xenograft rejection, the method comprising: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing a sequencing library from the extracted cell-free DNA, sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads, and quantifying the total amount of donor-derived cell-free DNA based on the sequencing reads; and (c) determining whether the amount of donor-derived cell-free DNA from the xenograft or a function thereof exceeds a cut-off threshold indicative of xenograft rejection or injury.

[0007] In one aspect, the present disclosure relates to a method for preparing an amplified DNA composition, the DNA being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA from the xenograft; (b) preparing an amplified DNA composition by multiplex targeted amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume to detect and quantify the amount of donor-derived cell-free DNA of an animal, wherein the target loci comprise a set of animal target loci and a set of human target loci; (c) determining whether the amount of donor-derived cell-free DNA of an animal or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0008] In one aspect, the present disclosure relates to a method for preparing an amplified DNA composition, the DNA being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing an amplified DNA composition by targeted amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise both human target loci and animal target loci, and sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amounts of both donor-derived cell-free DNA and xenotransplant recipient-derived cell-free DNA based on the sequencing reads, wherein the human loci and the animal loci are the same, and wherein the human reads and the animal reads are distinguished based on the inserted sequences; (c) determining whether the fraction of donor-derived cell-free DNA of an animal or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0009] In one aspect, the present disclosure relates to a method for preparing an amplified DNA composition, the DNA being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method being for determining xenograft rejection, the method comprising: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing an amplified DNA composition by targeted amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise one or more target loci indicative of xenograft rejection; and (c) determining the amount of the one or more target loci indicative of xenograft rejection, and determining whether the amount of the one or more target loci indicative of xenograft rejection or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0010] In one aspect, the present disclosure relates to a method for preparing a DNA composition, the DNA being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method being for determining xenograft rejection, wherein the DNA composition comprises one or more target loci indicative of xenograft rejection; and wherein the determining step further comprises determining the amount of the one or more target loci indicative of xenograft rejection, and determining whether the amount of the one or more target loci indicative of xenograft rejection or a function thereof exceeds a cut-off threshold indicative of xenograft rejection; wherein xenograft rejection is determined by a combination of (i) the amount of the one or more target loci indicative of xenograft rejection or a function thereof and (ii) the total amount or fraction of animal donor-derived cell-free DNA.

[0011] In one aspect, the present disclosure herein relates to a method for preparing a complementary DNA (cDNA) composition from RNA, the RNA being extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method being for determining xenograft rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; (b) preparing a cDNA sequencing library from the extracted RNA, and sequencing the cDNA sequencing library by high-throughput sequencing to obtain sequencing reads, and quantifying the total amount of donor-derived RNA based on the sequencing reads; (c) determining whether the total amount of donor-derived RNA from the xenograft or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0012] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA from the xenograft; (b) preparing an amplified cDNA composition by multiplex targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume to detect and quantify the amount of donor-derived RNA, wherein the target loci comprise a set of animal target loci and a set of human target loci; (c) determining whether the amount of donor-derived RNA target loci or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0013] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; (b) preparing an amplified cDNA composition by targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise both human target loci and animal target loci, and sequencing the amplified cDNA by high-throughput sequencing to obtain sequence reads, and quantifying the amounts of both donor-derived RNA and xenograft recipient-derived RNA based on the sequence reads, wherein the human loci and the animal loci are the same, and wherein the human reads and the animal reads are distinguished based on the inserted sequences; (c) determining whether the fraction of donor-derived RNA or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0014] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived cell-free RNA and recipient-derived RNA; (b) preparing an amplified cDNA composition by targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise one or more target loci indicative of xenograft rejection; and (c) determining the amount of the one or more target loci indicative of xenograft rejection, and whether the amount or a function of the one or more target loci indicative of xenograft rejection exceeds a cut-off threshold indicative of xenograft rejection.

[0015] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, wherein the composition comprises target loci indicative of xenograft rejection; and wherein the determining step further comprises determining the amount of the one or more target loci indicative of xenograft rejection, and determining whether the amount or a function of the one or more target loci indicative of xenograft rejection exceeds a cut-off threshold indicative of xenograft rejection, wherein xenograft rejection is determined by a combination of the amount or a function of the one or more target loci indicative of xenograft rejection and the total amount or a fraction of the animal donor-derived RNA.

[0016] In one aspect, the present disclosure relates to a method for preparing a protein composition from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting protein from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the protein comprises donor-derived protein from the xenograft and recipient-derived protein; (b) detecting and quantifying the amount of the donor-derived protein; (c) determining whether the amount or a function of the donor-derived protein exceeds a cut-off threshold indicative of xenograft rejection.

[0017] In one aspect, the present disclosure relates to a method for preparing a protein composition, the protein being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) extracting a protein from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the protein comprises a donor-derived protein and a recipient-derived protein from the xenograft; (b) detecting and quantifying the amount of one or more target proteins, wherein the one or more target proteins comprise both a recipient-derived protein and a donor-derived protein, and wherein the one or more target proteins indicate xenograft rejection; (c) determining whether the amount of the one or more target proteins or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0018] In one aspect, the present disclosure relates to a method for administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of a donor-derived protein as described herein; and (b) titrating the dose of the immunosuppressive therapy based on the amount of the donor-derived protein or a function thereof.

[0019] In one aspect, the present disclosure relates to a method for administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of one or more target proteins as described herein; and (b) titrating the dose of the immunosuppressive therapy based on the amount of the one or more target proteins or a function thereof.

[0020] In some embodiments, the methods herein further comprise longitudinally repeating steps (a) to (b) in the same xenotransplant recipient, and determining the longitudinal changes in the amount of the donor-derived protein, the donor-derived target protein or a function thereof and the longitudinal changes in the amount of the donor-derived protein, the target protein or a function thereof.

[0021] In some embodiments, the methods herein further comprise titrating the dose of the immunosuppressive therapy based on the longitudinal changes in the donor-derived protein, the donor-derived target protein or a function thereof.

[0022] In some embodiments, the protein is derived from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenotransplant recipient, and comprises extracting the protein from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenotransplant recipient.

[0023] In one aspect, the present disclosure relates to a method of administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (b) measuring the total amount of donor-derived cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dose of the immunosuppressive therapy based on the amount of cell-free DNA or a function thereof and the amount of donor-derived cell-free DNA or a function thereof.

[0024] In some embodiments, the amount of donor-derived cell-free DNA is measured by: extracting cell-free DNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification of the extracted DNA at 200-50,000 target loci in a single reaction volume; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads.

[0025] In some embodiments, the methods herein further comprise longitudinally repeating steps (a) to (b) for the same transplant recipient, and determining the longitudinal changes in the amount of cell-free DNA or a function thereof and the longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

[0026] In some embodiments, the methods herein further comprise titrating the dose of the immunosuppressive therapy based on the longitudinal changes in the total amount of cell-free DNA or a function thereof and the longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

[0027] In one aspect, the present disclosure relates to a method of administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of RNA in a blood, plasma, serum, or urine sample of the transplant recipient; (b) measuring the amount of donor-derived RNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dose of the immunosuppressive therapy based on the amount of cell-free DNA or a function thereof and the amount of donor-derived RNA or a function thereof.

[0028] In some embodiments, the amount of donor-derived RNA is measured by extracting RNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; preparing an amplified cDNA composition by multiplex targeted amplification of complementary DNA (cDNA) derived from the extracted RNA at 200 - 50,000 animal target loci in a single reaction volume to detect and quantify the amount of donor-derived RNA in the animal; sequencing the amplified cDNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of donor-derived RNA based on the sequencing reads.

[0029] In some embodiments, the methods herein further comprise longitudinally repeating steps (a) to (b) in the same transplant recipient, and determining the longitudinal changes in the amount of RNA or a function thereof and the longitudinal changes in the amount of donor-derived RNA or a function thereof.

[0030] In some embodiments, the methods herein further comprise titrating the dose of the immunosuppressive therapy based on the longitudinal changes in the total amount of RNA or a function thereof and the longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

[0031] In some embodiments, an internal control is added to the sample.

[0032] In some embodiments, the sequencing comprises shotgun whole-genome sequencing.

[0033] In some embodiments, the amount of DNA or RNA is measured by quantitative PCR, real-time PCR, digital PCR, or sequencing.

[0034] In some embodiments, the sequencing comprises next-generation whole-genome sequencing.

[0035] In some embodiments, wherein the amount of RNA or cell-free DNA is measured by using a microarray.

[0036] In some embodiments, the amount of donor-derived RNA or cell-free DNA is determined by using ratio-based and / or machine learning-artificial intelligence comparisons at single or multiple time points.

[0037] In some embodiments, the amount of RNA or cell-free DNA is measured by using molecular barcoding and microscopy imaging (such as NanoString )

[0038] In some embodiments, the target loci comprise single nucleotide polymorphisms (SNPs).

[0039] In some embodiments, the cut-off threshold is the estimated percentage of donor-derived cell-free DNA or RNA in total cell-free DNA or RNA or a function thereof.

[0040] In some embodiments, the cut-off threshold is proportional to the absolute concentration of donor-derived cell-free DNA or RNA concentration.

[0041] In some embodiments, step (b) includes amplifying 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 50-2000 pairs of forward and reverse PCR primers, for at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, at least 100 target loci, 2-10, 200-100, 50-500 or 50-2000 target loci.

[0042] In some embodiments, step (b) includes multiplex amplifying at least 100, at least 500, at least 1000, at least 2000, 10-1000, 100-10000, 50-50000, 500-20000 pairs of forward and reverse PCR primers, for at least 100, at least 500, at least 1000, at least 2000 target loci, 10-1000, 100-10000, 50-50000 or 500-20000 target loci.

[0043] In some embodiments, the RNA is cell-free RNA.

[0044] In some embodiments, the cell-free RNA is derived from exosomes or microvesicles.

[0045] In some embodiments, the RNA is small messenger RNA (mRNA).

[0046] In some embodiments, the RNA is small non-coding RNA (sncRNA).

[0047] In some embodiments, sncRNA includes microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA) or miscellaneous RNA (miscRNA).

[0048] In some embodiments, the methods herein further include using CRISPR-Cas to target and deplete contaminating or excessive nucleic acid species in the sample, thereby increasing the fraction of desired reads mapping to target loci of interest per sample and the sample throughput per sequencing run.

[0049] In some embodiments, the sample comprises whole blood or hemolyzed contaminated blood, serum or plasma samples, and wherein multiple guide RNAs are used to target multiple loci in the same reaction to deplete contaminated or excessive nucleic acids, thereby increasing the detection rate of the target loci.

[0050] In some embodiments, the contaminated or excessive nucleic acid species include hemoglobin mRNA, tRNA and rRNA, as well as miR-451, miR-144 and miR-486.

[0051] In some embodiments, the methods herein further comprise depleting adapter dimers, primer dimers and unwanted ligation products from the amplified nucleic acid composition comprising the target locus, thereby increasing the fraction of desired reads mapping to the target locus of interest per sample and the sample throughput per sequencing run.

[0052] In some embodiments, Cas9 / Cas12a is utilized to remove nucleic acid species after reverse transcription of RNA and prior to multiplex amplification.

[0053] In some embodiments, Cas9 / Cas12a is utilized to remove nucleic acid species after 1-10 cycles of multiplex amplification of the complementary DNA.

[0054] In some embodiments, the contaminated or excessive nucleic acid species are RNA, and wherein Cas13 is used to remove the contaminated or excessive RNA species from the sample.

[0055] In some embodiments, the xenograft recipient is a human subject.

[0056] In some embodiments, the xenograft recipient has received one or more xenograft organs selected from: pancreas, kidney, liver, heart, lung, intestine, thymus and uterus.

[0057] In some embodiments, the sample is obtained from the xenograft recipient less than 18 months after transplantation.

[0058] In some embodiments, logistic regression, random forest or decision tree machine learning analysis is used to determine the rejection risk of the xenograft recipient.

[0059] In some embodiments, the logistic regression, random forest or decision tree machine learning analysis further incorporates one or more parameters selected from: time after transplantation, age of the xenograft recipient and / or xenograft donor, sex of the xenograft recipient and / or xenograft donor.

[0060] In some embodiments, the xenograft is from a pig, primate, baboon, cow or dog.

[0061] In some embodiments, the cell-free DNA or RNA is derived from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine samples of the xenotransplant recipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 . Workflow of a test method for detecting cell-free DNA from an animal donor in a human plasma sample by using whole genome sequencing methods.

[0063] Figure 2 . Graph depicting the detection of cell-free DNA from an animal donor in a human plasma sample. The graph shows the linear relationship between the known amount of added cell-free DNA of animal origin and the amount measured using whole genome sequencing, thus demonstrating a proof of concept for the method of using whole genome sequencing to measure cell-free DNA of animal donor origin in a human plasma sample. DETAILED DESCRIPTION

[0064] The present disclosure relates to methods for determining and monitoring xenograft rejection in a human recipient based on whole genome sequencing of cell-free DNA or RNA from the blood, plasma, serum, or urine samples of the xenotransplant recipient. Alternatively, the present disclosure relates to methods for determining and monitoring xenograft rejection in a human recipient based on measuring proteins from the blood, plasma, serum, or urine samples of the xenotransplant recipient. In some embodiments, cellular DNA, RNA, or proteins are isolated from extracellular vesicles (EVs) that are isolated from the blood, plasma, serum, or urine samples of the xenotransplant recipient. The working examples presented herein show that the methods of the present disclosure can be used to detect nucleic acids of animal origin in human plasma samples.

[0065] Methods for determining and monitoring xenograft rejection based on measuring cell-free DNA.

[0066] In one aspect, the present disclosure relates to a method for preparing a DNA composition, wherein the DNA is derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenograft rejection. The method comprises: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing a sequencing library from the extracted cell-free DNA, sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads, and quantifying the total amount of animal donor-derived cell-free DNA based on the sequencing reads; (c) determining whether the amount of donor-derived cell-free DNA from the xenograft or a function thereof exceeds a cut-off threshold indicative of xenograft rejection or injury. In some embodiments, the extracted cell-free DNA is not amplified or pre-amplified prior to sequencing. In some embodiments, preparing the sequencing library comprises attaching adaptors to the extracted cell-free DNA, for example by ligation. In some embodiments, attaching the adaptors to the extracted cell-free DNA comprises end repair, adding adenosine to the cell-free DNA fragments, and subsequently blunt-end ligating to the cell-free DNA fragments. In some embodiments, the cell-free DNA fragments are repaired and filled in to generate blunt ends. In some embodiments, attaching the adaptors to the extracted cell-free DNA comprises blunt-end ligating the adaptors to the cell-free DNA fragments.

[0067] In one aspect, the present disclosure relates to a method for preparing an amplified DNA composition, wherein the DNA is derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenograft rejection. The method comprises: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA from the xenograft and recipient-derived cell-free DNA; (b) preparing an amplified DNA composition by multiplex targeted amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume to detect and quantify the amount of animal donor-derived cell-free DNA, wherein the target loci comprise a set of animal target loci and a set of human target loci; (c) determining whether the amount of animal donor-derived cell-free DNA or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0068] In one aspect, the present disclosure relates to a method for preparing an amplified DNA composition, wherein the DNA is derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenograft rejection. The method comprises: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing an amplified DNA composition by targeted amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise both human target loci and animal target loci, and sequencing the amplified DNA by high-throughput sequencing to obtain sequence reads, and quantifying the amounts of both donor-derived cell-free DNA and xenotransplant recipient-derived cell-free DNA based on the sequence reads, wherein the human loci and the animal loci are the same, and wherein human reads and animal reads are distinguished based on insertion sequences; (c) determining whether the fraction of animal donor-derived cell-free DNA or a function thereof exceeds a cut-off threshold indicative of xenograft rejection. As used herein, "insertion sequence" refers to any sequence that is different in the host human target locus compared to the same animal target locus.

[0069] In one aspect, the present disclosure relates to a method for preparing an amplified DNA composition, wherein the DNA is derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenograft rejection. The method comprises: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing an amplified DNA composition by targeted amplification of the extracted cell-free DNA at one or more target loci indicative of xenograft rejection in a single reaction volume; and (c) determining the amount of the one or more target loci indicative of xenograft rejection, and determining whether the amount of the one or more target loci indicative of xenograft rejection or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0070] In one aspect, the present disclosure relates to a method for preparing a DNA composition, wherein the DNA is derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenotransplant rejection, wherein the DNA composition comprises one or more target loci indicative of xenotransplant rejection; and wherein the determining step further comprises determining the amount of the one or more target loci indicative of xenotransplant rejection, and determining whether the amount of the one or more target loci indicative of xenotransplant rejection or a function thereof exceeds a cut-off threshold indicative of xenotransplant rejection; wherein xenotransplant rejection is determined by a combination of (i) the amount of the one or more target loci indicative of xenotransplant rejection or a function thereof and (ii) the total amount of cell-free DNA of animal donor origin or a fraction of cell-free DNA of animal donor origin.

[0071] In some embodiments, the cell-free DNA is derived from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenotransplant recipient.

[0072] A method for determining and monitoring xenotransplant rejection based on measuring RNA.

[0073] In one aspect, the present disclosure herein relates to a method for preparing a complementary DNA (cDNA) composition from RNA, wherein the RNA is extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenotransplant rejection, and the method comprises: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; (b) preparing a sequencing library of cDNA from the extracted RNA, and sequencing the cDNA by high-throughput sequencing to obtain sequencing reads, and quantifying the total amount of donor-derived RNA based on the sequencing reads; (c) determining whether the total amount of donor-derived RNA from the xenotransplant or a function thereof exceeds a cut-off threshold indicative of xenotransplant rejection. In some embodiments, the extracted RNA is not amplified or pre-amplified before sequencing. In some embodiments, preparing the sequencing library comprises attaching adapters to the cDNA, for example, by ligation. In some embodiments, the cDNA fragments are repaired and filled in to generate blunt ends. In some embodiments, the adapters are attached to the cDNA fragments by blunt-end ligation. In some embodiments, the cDNA is attached with adapters by sticky-end ligation to generate a sequencing library of cDNA.

[0074] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient for determining xenograft rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA from the xenograft; (b) preparing an amplified cDNA composition by multiplex targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume to detect and quantify the amount of donor-derived RNA, wherein the target loci comprise a set of animal target loci and a set of human target loci; (c) determining whether the amount of donor-derived RNA target loci or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0075] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient for determining xenograft rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; (b) preparing an amplified cDNA composition by targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise both human target loci and animal target loci, and sequencing the amplified cDNA by high-throughput sequencing to obtain sequence reads, and quantifying the amount of both donor-derived RNA and xenograft recipient-derived RNA based on the sequence reads, wherein the human loci and the animal loci are the same, and wherein the human reads and the animal reads are distinguished based on the inserted sequences; (c) determining whether the fraction of donor-derived RNA or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

[0076] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived cell-free RNA and recipient-derived RNA; (b) preparing an amplified cDNA composition by targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise one or more target loci indicative of xenograft rejection; and (c) determining the amount of the one or more target loci indicative of xenograft rejection, and whether the amount or a function of the one or more target loci indicative of xenograft rejection exceeds a cut-off threshold indicative of xenograft rejection.

[0077] In one aspect, the disclosure herein relates to a method for preparing an amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, wherein the composition comprises target loci indicative of xenograft rejection; and wherein the determining step further comprises determining the amount of the one or more target loci indicative of xenograft rejection, and determining whether the amount or a function of the one or more target loci indicative of xenograft rejection exceeds a cut-off threshold indicative of xenograft rejection, wherein xenograft rejection is determined by a combination of the amount or a function of the one or more target loci indicative of xenograft rejection and the total amount or a fraction of the animal donor-derived RNA.

[0078] In some embodiments, the RNA is derived from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenograft recipient.

[0079] A method for determining and monitoring xenograft rejection based on measuring proteins.

[0080] In one aspect, the present disclosure relates to a method for preparing a protein composition from a blood, plasma, serum, or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting proteins from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the proteins comprise donor-derived proteins and recipient-derived proteins from the xenograft; (b) detecting and quantifying the amount of donor-derived proteins; (c) determining whether the amount or a function of the donor-derived proteins exceeds a cut-off threshold indicative of xenograft rejection.

[0081] In one aspect, the present disclosure relates to a method for preparing a protein composition, the protein being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) extracting a protein from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the protein comprises a donor-derived protein and a recipient-derived protein from the xenograft; (b) detecting and quantifying the amount of one or more target proteins, wherein the one or more target proteins comprise both a recipient-derived protein and a donor-derived protein, and wherein the one or more target proteins indicate xenograft rejection; (c) determining whether the amount of the one or more target proteins or a function thereof exceeds a cut-off threshold indicating xenograft rejection.

[0082] In one aspect, the present disclosure relates to a method for preparing a protein composition, the protein being derived from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) extracting a protein from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the protein comprises a donor-derived protein and a recipient-derived protein from the xenograft; (b) detecting and quantifying the amount of the donor-derived protein; (c) determining whether the amount of the donor-derived protein or a function thereof exceeds a cut-off threshold indicating xenograft rejection.

[0083] In one aspect, the present disclosure relates to a method for preparing a protein composition, the protein being derived from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) extracting a protein from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the protein comprises a donor-derived protein and a recipient-derived protein from the xenograft; (b) detecting and quantifying the amount of one or more target proteins, wherein the one or more target proteins comprise both a recipient-derived protein and a donor-derived protein, and wherein the one or more target proteins indicate xenograft rejection; (c) determining whether the amount of the one or more target proteins or a function thereof exceeds a cut-off threshold indicating xenograft rejection.

[0084] In one aspect, the present disclosure relates to a method for administering an immunosuppressive therapy to a xenotransplant recipient, the method comprising: (a) measuring the amount of a donor-derived protein; and (b) titrating the dose of the immunosuppressive therapy based on the amount of the donor-derived protein or a function thereof.

[0085] In one aspect, the present disclosure relates to a method of administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of one or more target proteins; and (b) titrating the dose of the immunosuppressive therapy based on the amount of the one or more target proteins or a function thereof.

[0086] In some embodiments, the methods herein further comprise longitudinally repeating steps (a) to (b) in the same xenotransplant recipient, and determining longitudinal changes in the amount of donor-derived protein, donor-derived target protein or a function thereof and longitudinal changes in the amount of donor-derived protein, target protein or a function thereof.

[0087] In some embodiments, the methods herein further comprise titrating the dose of the immunosuppressive therapy based on the longitudinal changes in the amount of the donor-derived protein, donor-derived target protein or a function thereof.

[0088] Methods of measuring protein amounts include, but are not limited to, various sandwich, competitive or non-competitive assay formats to generate a signal related to the presence or amount of the protein analyte of interest. One agent for detecting the proteins of the present invention is, for example, an antibody capable of binding to the protein, preferably an antibody having a detectable label. The antibody can be polyclonal or, preferably, monoclonal. Intact antibodies or fragments thereof (e.g., Fab or F(ab')2) can be used. The term "label" is intended to encompass directly labeling the antibody by conjugating a detectable substance to the antibody, as well as indirectly labeling the antibody through reactivity with another directly labeled reagent.

[0089] Various formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, for example, enzyme immunoassays, radioimmunoassays, Western blot analysis, and ELISA. Numerous formats have been described and proposed for antibody arrays using antibodies. Such arrays typically comprise different antibodies specific for different proteins intended to be detected. For example, at least one hundred different antibodies are typically used to detect one hundred different protein targets, each antibody being specific for one target. In some embodiments, the amount of protein is measured using a mass spectrometry-based method. In a related aspect, the present invention provides an array comprising one or more supports carrying a plurality of ligands that specifically bind to a plurality of proteins. The plurality of proteins includes at least two, three, four, or five proteins determined to indicate transplant rejection. In some embodiments, the number of the plurality of proteins is less than 1000 or less than 100, more than 100, or more than 10, respectively. In some embodiments, the plurality of ligands are attached to a planar support or beads. In some embodiments, the ligands are different antibodies, and the different antibodies bind to different proteins among the plurality of proteins.

[0090] In some embodiments, the target protein is encoded by an RNA target disclosed elsewhere herein.

[0091] A sample comprising nucleic acid, and methods for obtaining the sample and extracting nucleic acid

[0092] The methods disclosed herein include extracting fragmented or intact RNA from a sample obtained from a xenograft recipient. In some embodiments, the xenograft recipient is a human subject and the xenograft donor is a pig. In some embodiments, the xenograft is from a pig, a primate, a baboon, a cow, or a dog.

[0093] In some embodiments, the xenograft recipient has received a plurality of transplanted organs selected from: pancreas, kidney, liver, lung, heart, intestine, thymus, or uterus. 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.

[0094] In some embodiments, the xenograft recipient has received one or more transplanted organs selected from: kidney, liver, heart, lung, pancreas, intestine, thymus, and uterus. In some embodiments, the xenograft recipient has received a kidney transplant. In some embodiments, the xenograft recipient has received a liver transplant. In some embodiments, the xenograft recipient has received a heart transplant. In some embodiments, the xenograft recipient has received a lung transplant. In some embodiments, the xenograft recipient has received a pancreas transplant. In some embodiments, the xenograft recipient has received an intestine transplant. In some embodiments, the xenograft recipient has received a thymus transplant. In some embodiments, the xenograft recipient has received a uterus transplant.

[0095] In some embodiments, the sample is obtained from the xenograft 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.

[0096] In some embodiments, the methods disclosed herein further comprise longitudinally measuring the amount of cell-free DNA, RNA, or protein in the same xenotransplant recipient; determining the longitudinal change in the amount of cell-free DNA, RNA, or protein. In some embodiments, the amount of cell-free DNA, RNA, or protein is the total amount of cell-free DNA, RNA, or protein derived from the donor organ.

[0097] In some embodiments, the xenotransplant recipient has received one or more organs from the same transplant donor. In some embodiments, the xenotransplant recipient has received one or more organs from multiple different transplant donors. In some embodiments, the xenotransplant recipient has received a simultaneous transplant of more than one organ.

[0098] The sample can be a body fluid sample, tissue, organ, or single cell. In some embodiments, the sample comprises blood, plasma, serum, CSF, or urine. In some embodiments, the sample is blood. In some embodiments, the sample is blood, plasma, or serum. In some embodiments, the sample can be extracellular vesicles derived from a body fluid sample such as blood, plasma, serum, CSF, or urine.

[0099] Nucleic acids and methods for extracting or enriching nucleic acids

[0100] The methods disclosed herein include extracting nucleic acids from a sample derived from a subject. The nucleic acids can be cell-free DNA, cellular DNA, DNA extracted from exosomes, 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). In some embodiments, the RNA can be cell-free RNA, cellular RNA, or exosomal RNA. In some embodiments, the RNA comprises small non-coding RNA (sncRNA). In some embodiments, the sncRNA comprises 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.

[0101] In some embodiments, nucleic acids are extracted by using size exclusion. In some embodiments, cell-free DNA or RNA is separated from cellular DNA or RNA based on size. In some embodiments, nucleic acids are separated by using affinity chromatography.

[0102] In some embodiments, nucleic acids are preferentially enriched. Preferential enrichment of nucleic acids is achieved 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 may involve selectively amplifying nucleic acid molecules corresponding to the locus. The method may involve removing nucleic acid molecules that do not correspond to the locus. The method may 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 may refer to the average enrichment level of all loci in the set of loci.

[0103] Amplification refers to a method of increasing the copy number of nucleic acid molecules. Selective amplification may 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 may 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.

[0104] 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 adaptor or "ligation adaptor" 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 adaptor 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 those DNA molecules corresponding to a set of loci in a DNA mixture.

[0105] Specific nucleic acids can also be enriched by using hybridization capture. In some embodiments, preferentially enriching RNA at multiple biomarkers includes: obtaining a set of hybridization capture probes; hybridizing the hybridization capture probes to the RNA in the sample; and physically separating the hybridized RNA from the unhybridized RNA in the RNA sample. In some embodiments, preferentially enriching sncRNA (such as miRNA) at multiple biomarkers includes: obtaining a set of hybridization capture probes; hybridizing the hybridization capture probes to the miRNA in the sample; and physically separating the hybridized miRNA from the unhybridized RNA in the RNA sample. In some embodiments, preferentially enriching a preselected mRNA includes: obtaining a set of hybridization capture probes; hybridizing the hybridization capture probes to the mRNA in the sample; and physically separating the hybridized mRNA from the unhybridized RNA in the RNA sample.

[0106] In some embodiments, in the methods disclosed herein, DNA is preferentially enriched at target loci or biomarkers.

[0107] 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 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 the 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 un-diseased tissue or cell sample from a subject with or suspected of having a disease or compared to a cell or tissue sample from a subject or 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.

[0108] In some embodiments, one or more biomarkers can be one or a set 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 a genome, underexpression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or a genome, alternative production of a splice variant of a gene or a genome, gene copy number variant (CNV) (e.g., DNA double minute), nucleic acid modification (e.g., methylation, acetylation, and phosphorylation), single nucleotide polymorphism (SNP), chromosomal rearrangement (e.g., inversion, deletion, and duplication), and mutation (insertion, deletion, duplication, missense, nonsense, synonymous, or any other nucleotide change) of a gene or a genome. In many cases, these mutations 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.

[0109] In some embodiments, preferentially enriching DNA in a sample at a plurality of 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 probe with DNA from the sample; using a DNA polymerase to fill the gap between the ends of the hybridized probe; circularizing the pre-circularized probe; and amplifying the circularized probe.

[0110] In some embodiments, preferentially enriching DNA at a plurality of 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 probe with DNA from the first sample; using a DNA polymerase to fill the gap between the ends of the ligation-mediated PCR probe; ligating the ligation-mediated PCR probe; and amplifying the ligated ligation-mediated PCR probe.

[0111] In some embodiments, preferentially enriching DNA at multiple polymorphic loci comprises: obtaining a plurality of hybridization capture probes that target the polymorphic loci; hybridizing the hybridization capture probes to DNA in a sample; and physically removing some or all of the unhybridized DNA from the first DNA sample.

[0112] In some embodiments, the hybridization capture probes are designed to hybridize to regions flanking but not overlapping the polymorphic loci. In some embodiments, the hybridization capture probes are designed to hybridize to regions flanking but not overlapping the polymorphic loci, 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 the polymorphic loci, and wherein the plurality of hybridization capture probes comprises 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.

[0113] In some embodiments, preferentially enriching DNA at multiple 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.

[0114] 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 located upstream of the internal forward primer; 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 located immediately downstream of the internal reverse primer; hybridizing the first primer to the DNA; and amplifying the DNA using polymerase chain reaction.

[0115] In some embodiments, the method further comprises: 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 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 in the polymorphic locus, and wherein the external forward primers are designed to hybridize to a DNA region located upstream of the internal forward primer; hybridizing the first primer to the DNA; and amplifying the DNA using polymerase chain reaction.

[0116] In some embodiments, preparing the first sample further comprises attaching a universal linker to the DNA in the first sample and amplifying the DNA in the first sample 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%.

[0117] In some embodiments, the DNA amplification is performed in one or more separate reaction volumes, and wherein each separate reaction volume contains 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.

[0118] In some embodiments, preparing the sample further comprises dividing the sample into multiple portions, and wherein the DNA in each portion is preferably enriched at a subset of multiple 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 multiple primers. In some embodiments, the internal primers contain regions designed to hybridize upstream or downstream of the targeted polymorphic locus and optionally contain universal primer sequences designed to allow PCR amplification. In some embodiments, at least some of the primers further contain random regions that are different for each individual primer molecule. In some embodiments, at least some of the primers further contain molecular barcodes.

[0119] In some embodiments, the method comprises: (a) performing multiplex polymerase chain reaction (PCR) on a nucleic acid sample containing a target locus in a single reaction volume using (i) at least 1,000 different primer pairs or (ii) at least 1,000 target-specific primers and universal or tag-specific primer pairs to simultaneously amplify at least 1,000 different target loci, thereby generating an amplification product containing target amplicons; and (b) sequencing the amplification product. In some embodiments, the method does not include the use of a microarray.

[0120] In some embodiments, the method comprises (a) performing multiplex polymerase chain reaction (PCR) on a cell-free DNA sample containing a target locus using (i) at least 1,000 different primer pairs, or (ii) at least 1,000 target-specific primers and universal or tag-specific primer pairs in a single reaction volume to simultaneously amplify at least 1,000 different target loci, thereby generating an amplification product containing target amplicons; and b) sequencing the amplification product. In some embodiments, the method does not include the use of a microarray.

[0121] In some embodiments, mRNA is isolated by using a probe that hybridizes to the polyA tail of the mRNA molecule.

[0122] After blood draw and before 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 cell-free DNA (cfDNA) and background noise, thereby distorting the thd% of the detected dd-cfDNA. To reduce such background noise, and based on the observation that dd-cfDNA is typically shorter than DNA fragmented from transplant recipient blood cells, two special 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 noise (e.g., before applying multiplex PCR).

[0123] Target genes and loci and protein targets

[0124] The nucleic acid may include biomarkers indicative of an immune response or various diseases or conditions as described elsewhere herein. In some embodiments, the target locus includes one or more different sets of target loci. In some embodiments, the target locus contains a set of human target loci and a set of animal target loci, wherein the set of human target loci is different from the set of animal target loci. In some embodiments, the set of human target loci and the set of animal target loci are the same, and one or more human target loci can be distinguished from the corresponding animal loci by insertion sequences.

[0125] In some embodiments, the method includes extracting fragmented or intact mRNA from a sample derived from a xenotransplant recipient, wherein the extracted mRNA contains donor and / or recipient-derived mRNA, and wherein the mRNA contains 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. In some specific embodiments, sncRNA (such as miRNA) biomarkers indicate an increased immune response or a decreased immune response.

[0126] In some embodiments, the methods of the present disclosure include preselecting RNA target molecules. In some embodiments, the RNA target molecules include RNA species known to be related to assessing organ health. In some embodiments, the present disclosure provides methods for identifying RNA target molecules related to assessing organ health.

[0127] In some embodiments, the methods disclosed herein further include preferentially enriching RNA at multiple biomarkers indicative of xenograft rejection. In some embodiments, the RNA biomarkers indicate an increased immune response or a decreased immune response.

[0128] In some embodiments, the biomarker comprises a single nucleotide polymorphism (SNP) locus.

[0129] Sample and method for isolating nucleic acid from a sample

[0130] 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.

[0131] In some embodiments, the nucleic acid sample comprises RNA, such as cfRNA, cellular RNA, cytoplasmic RNA, coding cytoplasmic RNA, non-coding cytoplasmic RNA, mRNA, miRNA, mitochondrial RNA, rRNA, or tRNA. 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 human nucleic acid. In some embodiments, the target locus is a segment of human nucleic acid present in the human 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.

[0132] In some embodiments, the method comprises isolating or purifying DNA and / or RNA. 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 or RNA. In some embodiments, the preparation of DNA or RNA can involve amplification, fractionation, purification by chromatography, liquid fractionation, separation, 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 of isolating RNA, DNase (such as DNase I from Invitrogen, Carlsbad, Calif., USA) is used to degrade DNA. In some embodiments, RNEASY TMThe mini kit (Qiagen) isolates RNA according to the manufacturer's protocol. In some embodiments, MIRVANA is used TM The PARIS kit (Ambion, Austin, Tex., USA) isolates small RNA molecules according to the manufacturer's protocol (Gu et al., J. Neurochem. 122:641–649, 2012, which is incorporated herein by reference in its entirety). Nanovue (GE Healthcare, Piscataway, N.J., USA) can be optionally used to determine the concentration and purity of RNA, and the RNA integrity can be optionally measured by using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA) (Gu et al., J. Neurochem. 122:641–649, 2012, which is incorporated herein by reference in its entirety). In some embodiments, TRIZOL or RNALATER TM (Ambion) is used to stabilize RNA during storage.

[0133] In some embodiments, adapters are added to construct a sequencing library. Before ligation, the sample DNA can be blunt-ended and then a single adenosine base is added to the 3' end. In some embodiments, the ligation of the adapter to the nucleic acid is a sticky-end ligation. Before 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, a ligation kit present in the AGILENT SURESELECT TM kit is used for adapter ligation.

[0134] 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 the target locus. In some embodiments, the amplified DNA is sequenced (such as using an ILLUMINAIIGAX TM or HiSeq sequencer for sequencing). 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).

[0135] In some embodiments, miRNAs can be separated from RNA fragments caused by degradation because the degraded RNAs lose the phosphorylated groups at the ends. miRNAs retain the phosphorylated groups at the ends. An adaptor can be ligated to the phosphorylated miRNA ends, but the adaptor will not ligate to unphosphorylated RNA species such as degraded mRNAs. The adaptor can contain a sequence that allows primer binding to assist in selectively generating complementary DNA (cDNA) by reverse transcription.

[0136] As a non-limiting example, the locus can be a single nucleotide polymorphism, an intron, or an exon. In some embodiments, the 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 free-floating DNA in a blood, serum, or plasma sample (e.g., circulating cell-free tumor DNA or circulating cell-free fetal DNA). In these embodiments, the sample is typically 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 free-floating DNA from blood. Plasma samples can be stored at -80 °C until DNA is extracted using, for example, the QIAAMP TM DNA Mini Kit (Qiagen, Hilden, Germany) (e.g., Hamakawa et al., Br J Cancer. 2015;112:352-356). However, the sample can be derived from other sources, and nucleic acid molecules from any organism can be used in this method. In some embodiments, DNA from bacteria and / or viruses can be used to analyze true sequence variants within a mixed population, especially in environmental and biodiversity sampling.

[0137] 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, NEXTFLEX TM Cell Free Kit (Bioo Scientific, Austin, Tex.) or Natera Library Prep Kit (Natera, San Carlos, Calif.). Such kits are typically modified to include adaptors customized for the amplification and sequencing steps in the methods provided herein. Commercially available kits (such as those present in Agilent SURESELECT TMAdapter ligation was carried out using a ligation kit (Agilent, Santa Clara, Calif.) in the kit.

[0138] 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, nucleic acids initially isolated from a tissue, fluid, or cultured cell may be much longer than the sample nucleic acid molecules processed using the methods herein. As discussed herein, for example, such initially isolated nucleic acid molecules can be fragmented prior to use in the methods herein to generate nucleic acid segments. 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.

[0139] In some embodiments, the entire sample nucleic acid molecule is the sample nucleic acid segment. For example, in certain embodiments, where an adaptor 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 adaptor (such as a universal adaptor 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 where the adaptor is attached to the sample nucleic acid molecule as part of a primer that targets a binding site internal to the end of the sample nucleic acid molecule, a part of the sample nucleic acid molecule can be the sample nucleic acid segment that is 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.

[0140] 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, while 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 the gene of interest) can be enriched prior to performing the methods provided herein.

[0141] Removing contamination

[0142] Biological samples may contain a large amount of contaminating nucleic acids, which may make the detection of target nucleic acids difficult, reduce the quality of nucleic acid libraries, and / or reduce the throughput of assays. For example, blood cells within a blood sample may burst and shed nucleic acids into the sample, resulting in the sample potentially containing a large amount of irrelevant nucleic acids from red blood cells. In one aspect of the present disclosure, the genome editing CRISPR-Cas system can be used to remove contaminating nucleic acids. CRISPR-Cas technology allows for simple, flexible, and relatively inexpensive cleavage of DNA and / or RNA in a targeted sequence-specific manner. The components required for in vitro digestion include a Cas effector protein and a target-specific guide RNA (gRNA).

[0143] An effective CRISPR-Cas complex must have several characteristics. First, the gRNA must have a spacer element that is complementary to the target nucleic acid. This complementary target nucleic acid sequence is referred to as the protospacer. Additionally, located immediately downstream of the spacer (for Cas9, for Cas12a it is located immediately upstream of the spacer), there must be a protospacer adjacent motif (PAM). For Cas9, the motif can be NGG, where N is any nucleotide, and for Cas12a, the PAM motif can be TTTV, where V is any nucleotide other than T. Proper complex formation also requires the scaffold of the gRNA. However, since this is conserved for all gRNAs of the same Cas enzyme, IDT has standardized this for Cas9 and created a separate "tracrRNA" that anneals to a shorter "crRNA" to form the complete gRNA.

[0144] A variety of CRISPR Cas systems have been developed. The CRISPR Cas9 system is the first and best-characterized single-protein CRISPR effector and is subdivided into types II-A, II-B, and II-C. Cas9 creates blunt double-stranded DNA breaks, which can then be repaired by non-homologous end joining or homologous recombination with donor template DNA to create site-specific edits. Type II-A Cas9 generally has high genome editing efficiency, but off-target cleavage at unexpected genomic sites can be a disadvantage. Variants have been designed to overcome these limitations, and type II-C Cas9 tends to naturally have higher fidelity. Cas9 uses guide spacers that are 18 - 24 nucleotides (nt) in length. The total length of the guide spacer for Cas9 can be approximately 100 nt. The PAM sequence can be 3-NGG (SpCas9), 3-NNGRRT (SaCas9), 3-NNNNGATT (NmCas9). Cas9 produces blunt-ended dsDNA breaks. Mutant Cas9 enzymes are also commercially available, which create single-strand nicks in the DNA on one strand of the target, as well as non-cutting mutants that only bind the target.

[0145] Cas12 belongs to type V CRISPR-Cas, which includes subtypes V-A and V-B (also known as Cpf1 (type V-A) or C2c1 (type V-B)), among other subtypes. Cas12 is a compact and efficient enzyme that creates staggered cuts in dsDNA and thus creates 3- to 5-base overhangs. The guide spacer of Cas12 has a length of 18 - 25 nt, and the total guide length is 42 - 44 nt. The Cas12 PAM sequence can be 5-TTTN (FnCas12a). Cas12 processes its own guide RNA, thus enhancing multiplexing capabilities. Cas12 has also been engineered as a platform for epigenome editing, and it has recently been found that Cas12a can indiscriminately shred single-stranded DNA once activated by a target DNA molecule that matches its spacer sequence.

[0146] Cas13 is a type VI CRISPR-Cas, which includes subtypes VI-A, VI-B, VI-C, and VI-D, and is also known as C2c2 (VI-A) or CasRx (type VI-D). Cas13 targets RNA rather than DNA. The guide spacer of Cas13 has a length of 22-30 nt, and the total guide length is 52-66 nt. The Cas13 PAM sequences include 3-H (LshCas13a), 5-D, and 3-NAN or NNA (BzCas13b), and there is none (RfCas13d). Once Cas13 is activated by an ssRNA sequence complementary to its crRNA spacer, Cas13 exhibits non-specific RNase activity, destroying all nearby RNAs regardless of their sequences. This property has been used for accurate diagnosis in vitro. These systems can also be used for efficient, reusable, and specific RNA knockout or RNA sequence editing in mammalian cells.

[0147] The methods disclosed herein can include using any CRISPR-Cas system to deplete contaminating or excessive nucleic acids in a biological sample or library. For example, Cas9 and / or Cas12 can be used to remove DNA species in whole blood or hemolyzed contaminated blood, serum, or plasma samples, thereby increasing the sensitivity for detecting target loci. On the other hand, Cas enzymes targeting RNA species can be used directly. For example, Cas13 can be used to remove contaminating RNA species from a biological sample prior to library preparation.

[0148] Cas enzymes can be given a mixture of different gRNAs to allow targeting of many different loci in the same reaction. In some embodiments, 1 gRNA is used to target a locus. In some embodiments, 1 - 5 gRNAs are used to target 1 - 5 loci in the same reaction. In some embodiments, 1 - 10 gRNAs are used to target 1 - 10 loci in the same reaction. In some embodiments, 1 - 100 gRNAs are used to target 1 - 100 loci in the same reaction. In some embodiments, 1 - 5000 gRNAs are used to target 1 - 5000 loci in the same reaction. In some embodiments, 10 - 5000 gRNAs are used to target 10 - 5000 loci in the same reaction. In some embodiments, 100 - 5000 gRNAs are used to target 100 - 5000 loci in the same reaction. In some embodiments, 1000 - 50000 gRNAs are used to target 1000 - 50000 loci in the same reaction. In some embodiments, 10000 - 5000 gRNAs are used to target 10000 - 50000 loci in the same reaction. In some embodiments, multiple gRNAs are used to target multiple target loci in the same reaction. In some embodiments, at least 10 gRNAs are used to target at least 10 loci in the same reaction. In some embodiments, at least 20 gRNAs are used to target at least 20 loci in the same reaction. In some embodiments, at least 50 gRNAs are used to target at least 50 loci in the same reaction. In some embodiments, at least 100 gRNAs are used to target at least 100 loci in the same reaction. In some embodiments, at least 500 gRNAs are used to target at least 500 loci in the same reaction. In some embodiments, at least 1000 gRNAs are used to target at least 1000 loci in the same reaction. In some embodiments, at least 2000 gRNAs are used to target at least 2000 loci in the same reaction. In some embodiments, at least 3000 gRNAs are used to target at least 3000 loci in the same reaction. In some embodiments, at least 4000 gRNAs are used to target at least 4000 loci in the same reaction. In some embodiments, at least 5000 gRNAs are used to target at least 5000 loci in the same reaction. In some embodiments, at least 10000 gRNAs are used to target at least 10000 loci in the same reaction. In some embodiments, at least 20000 gRNAs are used to target at least 20000 loci in the same reaction.

[0149] Non-targeted miRNA analysis can be performed by ligating the miRNA to an adaptor as described above. The adaptor can provide an NGG position adjacent to the miRNA to allow Cas9 cleavage. When an undesired sequence is ligated to the adaptor, a Cas9-gRNA complex complementary to this undesired sequence will be introduced. This will result in the removal of the adaptor and prevent PCR amplification. This step is performed on double-stranded reverse transcribed DNA.

[0150] In some embodiments, a pilot sequencing run can be used to inform which targets need to be removed from future sequencing runs. This will allow assay developers to focus only on the miRNAs of interest by designing gRNAs that target the most common non-concerned or contaminating small RNA fragments actually observed in the specific sample type used for the assay. This may include highly abundant miRNAs or small fragments of ribosomal or messenger RNAs that may cause background noise.

[0151] In some embodiments, the Cas enzyme can be removed by using heat-labile Proteinase K (NEB P8111S) because the Cas complex is relatively long-lived and can interfere with downstream applications.

[0152] Although this method can also be used in targeted miRNA applications, it is preferred to improve / remove bad primers before the assay rather than trying to remove them after amplification using CRISPR-Cas.

[0153] In some embodiments, CRISPER-Cas-mediated contaminant species removal can also be used in mRNA applications. In some embodiments, the CRISPR-Cas method is used to remove contaminating mRNA species that are less than 20 nucleotides in length. When the contaminating mRNA species are less than 20 nucleotides in length, a method similar to the method proposed for miRNAs can be used to design assays to specifically target the observed sequences. For example, this may be particularly useful if barcoding primer dimers consume most of the sequencing reads.

[0154] In some embodiments, the CRISPR-Cas method is used to remove contaminating mRNA species that are greater than 20 nucleotides in length. In some embodiments, the CRISPR-Cas method is used in combination with a "tag and capture" method to remove contaminating mRNA species that are greater than 20 nucleotides in length.

[0155] In some embodiments, prior to amplification, cDNA derived from miRNA or mRNA is subjected to a CRISPR-Cas method to remove contaminating species. In some embodiments, cDNA derived from miRNA or mRNA is subjected to a CRISPR-Cas method to remove contaminating species, and wherein the cDNA is amplified for 1 to 5 cycles, 1 to 10 cycles, or 1 - 15 cycles. In some embodiments, prior to CRISPR-Cas-mediated removal of contaminating species, the cDNA is amplified no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles.

[0156] In some embodiments, contaminating or excessive species include hemoglobin mRNA, tRNA, rRNA, and miRNAs such as miR-451, miR-144, and miR-486, thereby increasing the fraction of desired reads mapped to a target locus of interest per sample and the sample throughput per sequencing run.

[0157] In some embodiments, the method further comprises depleting adapter dimers, primer dimers, and undesired ligation products from the amplified nucleic acid composition comprising the target locus, thereby increasing the fraction of desired reads mapped to a target locus of interest per sample and the sample throughput per sequencing run.

[0158] Method for identifying biomarkers of interest for healthy organ transplantation

[0159] Text mining databases to identify biomarkers known to be of interest for transplantation health. Identify biomarkers associated with the health of transplanted organs and look for common features relative to a randomly selected set of biomarkers. Artificial intelligence can be used for text mining and to predict biomarkers known to be of interest for transplant rejection and organ health.

[0160] Combining the measurement of cell-free DNA with the measurement of RNA to determine and / or monitor transplant rejection

[0161] In some embodiments, the methods herein further comprise: (i) measuring the amount of donor-derived cell-free DNA in a sample obtained from the xenograft recipient, extracting cell-free DNA from the sample obtained from the xenograft recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (ii) performing targeted amplification of the extracted DNA at 50 - 50,000 target loci in a single reaction volume; (iii) sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads, determining xenograft rejection based on whether the amount of donor-derived cell-free DNA or a function thereof exceeds a cut-off threshold indicative of cell-free DNA amount in xenograft rejection, wherein xenograft rejection is determined based on whether the amount of donor-derived RNA and the amount of donor-derived cell-free DNA or a function thereof exceed a cut-off threshold indicative of xenograft rejection. The combination of the amounts of RNA and cfDNA in a sample obtained from a xenograft recipient can serve as a biomarker for rejection and as a biomarker for the net state of immunosuppression. In some embodiments, the combination of the amount of an mRNA target selected from a set of preselected targets and the amount of cfDNA in a sample indicates transplant rejection. In another aspect, the rejection risk of a transplant recipient can be determined based on the amount of donor-derived RNA and / or the amount of cell-free DNA. In another aspect, the rejection risk of a transplant recipient can be determined based on the amount of donor-derived mRNA and / or the amount of cell-free DNA. In another aspect, the rejection risk of a transplant recipient can be determined based on the amount of donor-derived miRNA and / or the amount of cell-free DNA.

[0162] Determine the rejection risk of a transplant recipient

[0163] In some embodiments, a logistic regression, random forest, or decision tree machine learning analysis is used to determine the rejection risk of a xenograft recipient. In some embodiments, the machine learning analysis incorporates the amount of donor-derived RNA or a function thereof in a sample of the transplant recipient as a parameter. In some embodiments, the machine learning analysis incorporates the number of reads of donor-derived RNA or a function thereof as a parameter. In some embodiments, the machine learning analysis incorporates the estimated percentage of donor-derived RNA in total RNA as a parameter. In some embodiments, the machine learning analysis incorporates the amount of cell-free DNA, the number of reads of cell-free DNA, or the estimated percentage of cell-free DNA in total cell-free DNA in a sample of the transplant recipient as a parameter. In some embodiments, the machine learning analysis incorporates the total amount of multiple proteins derived from the xenograft. In some embodiments, the machine learning analysis further incorporates the amount of total cell-free DNA or a function thereof in a sample of the transplant recipient as a parameter. In some embodiments, the machine learning analysis further incorporates the number of reads of total cell-free DNA or a function thereof as a parameter.

[0164] Machine learning can be used to solve the problem of rejection and non-rejection. Machine learning is disclosed in WO2020 / 018522, titled "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 or RNA in the blood sample.

[0165] In some embodiments, the cut-off threshold is expressed as a percentage of dd-cfDNA (dd-cfDNA%) in the blood sample. 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.

[0166] 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 genome copies / plasma volume, cell-free DNA 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), HLA alleles or epitope mismatches of the recipient and / or donor, Banff classification of renal allograft pathology, and cause and surveillance or protocol biopsies.

[0167] In some embodiments, when the amount of dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 50% sensitivity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 60% sensitivity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of total cfDNA in the blood sample and a 95% confidence interval, the method has at least 70% sensitivity in identifying acute rejection (AR) relative to non-AR. In some embodiments, when the amount of dd-cfDNA is higher than the cut-off threshold scaled or adjusted 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 amount of dd-cfDNA is higher than the cut-off threshold scaled or adjusted 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 amount of dd-cfDNA is higher than the cut-off threshold scaled or adjusted 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 amount of dd-cfDNA is higher than the cut-off threshold scaled or adjusted 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.

[0168] In some embodiments, when the amount of the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the total cfDNA in the blood 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 the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the total cfDNA in the blood 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 the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the 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 the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the 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 the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the total cfDNA in the blood 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 the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the 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 the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the 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 the dd-cfDNA is higher than the cut-off threshold scaled or adjusted according to the amount of the 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.

[0169] Some embodiments of the present invention relate to a method for quantifying the amount of donor-derived cell-free DNA in a biological sample of a transplant recipient, the method comprising: a) isolating cell-free DNA from the biological sample of the transplant recipient, wherein the isolated cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA, and wherein a first tracer DNA composition is added before or after the isolation of the cell-free DNA; b) performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs; c) sequencing the amplification products by high-throughput sequencing to generate sequencing reads; and d) quantifying the amount of donor-derived cell-free DNA and the amount of total cell-free DNA, wherein the amount of total cell-free DNA is quantified using the sequencing reads derived from the first tracer DNA composition.

[0170] Some embodiments use a fixed or non-fixed threshold of donor DNA per plasma volume, adjusted or scaled as described herein. The threshold can be determined by establishing an algorithm using a training dataset to maximize performance. It can also consider other data such as the patient's weight, age, or other clinical factors.

[0171] In some embodiments, the method further comprises determining that transplant rejection has occurred or is likely to occur using the amount of donor-derived cell-free DNA. In some embodiments, the amount of donor-derived cell-free DNA is compared to a cut-off threshold to determine that transplant rejection has occurred or is likely to occur, wherein the cut-off threshold is adjusted or scaled according to the amount of total cell-free DNA. In some embodiments, the cut-off threshold is a function of the number of reads of donor-derived cell-free DNA.

[0172] In some embodiments, the method comprises applying a scaled or dynamic threshold metric that takes into account the total cfDNA amount in the sample to more accurately assess transplant rejection. In some embodiments, the method further comprises flagging the sample if the amount of total cell-free DNA is above a predetermined value. In some embodiments, the method further comprises flagging the sample if the amount of total cell-free DNA is below a predetermined value.

[0173] RNA, DNA, or protein can be extracted from a sample of a transplant recipient, wherein the sample includes blood, plasma, serum, cerebrospinal fluid (CSF), or urine.

[0174] In some embodiments, the machine learning analysis further incorporates the time after transplantation 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 gender of the transplant recipient and / or the transplant donor as a parameter.

[0175] In some embodiments, the risk of rejection of the transplant recipient is determined with a sensitivity of at least 0.81, or at least 0.82, or at least 0.83, or at least 0.84, or at least 0.85, or at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90. In some embodiments, the risk of rejection of the transplant recipient is determined with a specificity of at least 0.81, or at least 0.82, or at least 0.83, or at least 0.84, or at least 0.85, or at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90. In some embodiments, the risk of rejection 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.

[0176] Method for measuring the amount of nucleic acid

[0177] In some embodiments, the amount of RNA is measured by quantitative PCR. In some embodiments, the amount of RNA is measured by real-time PCR. In some embodiments, the amount of RNA is measured by digital PCR. In some embodiments, the amount of RNA is measured by sequencing such as high-throughput sequencing, next-generation sequencing, or sequencing by synthesis.

[0178] In some embodiments, the amount of donor-derived nucleic acid (e.g., RNA and / or DNA) is determined by using ratio-based and / or machine learning-artificial intelligence comparisons at single or multiple time points. In some embodiments, the amount of donor-derived mRNA is determined by using ratio-based and / or machine learning-artificial intelligence comparisons at single or multiple time points. In some embodiments, the amount of donor-derived miRNA is determined by using ratio-based and / or machine learning-artificial intelligence comparisons at single or multiple time points.

[0179] In some embodiments, the amount of RNA or cell-free DNA is measured by a quantitative PCR method. In some embodiments, the amount of mRNA is measured by a quantitative PCR method. In some embodiments, the amount of miRNA is measured by a quantitative PCR method. In some embodiments, the quantitative PCR method includes real-time PCR or digital PCR.

[0180] In some embodiments, the amount of mRNA or cell-free DNA is measured by large-scale multiplex PCR (mmPCR) to obtain amplicons containing biomarkers, and the amplicons are sequenced.

[0181] In some embodiments, the amount of nucleic acid (e.g., mRNA, miRNA, or cell-free DNA) is measured by using a microarray.

[0182] In some embodiments, the amount of nucleic acid (e.g., mRNA, miRNA, or cell-free DNA) is measured by using molecular barcodes and microscopy imaging (such as NanoString ).

[0183] In some embodiments, the amount of donor-derived RNA is measured by: extracting RNA from a blood, plasma, serum, cerebrospinal fluid (CSF), or urine sample of a transplant recipient, wherein the extracted RNA contains donor-derived RNA and recipient-derived RNA; performing targeted amplification of the extracted RNA at 100 - 50,000 target loci in a single reaction volume; sequencing the amplified RNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of donor-derived RNA based on the sequencing reads. In some embodiments, the amplification of RNA includes performing reverse transcriptase to obtain complementary DNA (cDNA).

[0184] In some embodiments, the amount of donor-derived cell-free DNA is measured by: extracting cell-free DNA from a blood, plasma, serum, cerebrospinal fluid (CSF), or urine sample of a transplant recipient, wherein the extracted cell-free DNA contains donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification of the extracted DNA 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 amount of donor-derived cell-free DNA based on the sequencing reads.

[0185] 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.

[0186] In some embodiments, the amount of nucleic acid is measured by targeted amplification. In some embodiments, the amount of a specific mRNA 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.

[0187] In some embodiments, target amplification includes nested PCR. In some embodiments, the primers for target 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, target 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.

[0188] In some embodiments, the method further includes attaching a tag to the amplification product 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 RNA 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 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.

[0189] 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. A tracer can be added to a biological sample derived from a human subject to assist in estimating the amount of total RNA or cfDNA in the sample. It can also be added to a reaction mixture other than the biological sample itself.

[0190] For determining the cut-off threshold for transplant rejection

[0191] In some embodiments, the cut-off threshold is the estimated percentage of donor-derived RNA in total RNA or a function thereof. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% RNA (such as mRNA or miRNA). In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% cell-free DNA or a combination of cell-free DNA and RNA. 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.

[0192] In some embodiments, the cut-off threshold is the estimated percentage of donor-derived RNA in total RNA or a function thereof. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% RNA. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% cell-free DNA or a combination of cell-free DNA and RNA. 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.

[0193] In some embodiments, the cut-off threshold is the estimated percentage of donor-derived mRNA in the total mRNA or a function thereof. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% RNA. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% cell-free DNA or a combination of cell-free DNA and mRNA. 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.

[0194] In some embodiments, the cut-off threshold is the estimated percentage of a preselected donor-derived mRNA target in the total mRNA or a function thereof. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% RNA. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% cell-free DNA or a combination of cell-free DNA and the preselected mRNA target. 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.

[0195] In some embodiments, the cut-off threshold is the estimated percentage of donor-derived protein in the total protein or a function thereof. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% protein. In some embodiments, the cut-off threshold is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% cell-free DNA or a combination of cell-free DNA and protein. 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.

[0196] In some embodiments, the cut-off threshold is proportional to the absolute donor-derived RNA concentration. In some embodiments, the cut-off threshold is the copy number of donor-derived RNA or a function thereof. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of RNA. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of RNA per unit volume of blood sample. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of RNA per unit volume of blood sample multiplied by the body weight, BMI, or blood volume of the transplant recipient.

[0197] In some embodiments, the cut-off threshold is proportional to the absolute donor-derived RNA concentration. In some embodiments, the cut-off threshold is the copy number of donor-derived RNA or a function thereof. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of RNA. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of RNA per unit volume of blood sample. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of RNA per unit volume of blood sample multiplied by the body weight, BMI, or blood volume of the transplant recipient.

[0198] In some embodiments, the cut-off threshold is proportional to the absolute donor-derived protein concentration. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of protein. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of protein per unit volume of blood sample. In some embodiments, the cut-off threshold is expressed as the amount or absolute amount of protein per unit volume of blood sample multiplied by the body weight, BMI, or blood volume of the transplant recipient.

[0199] In some embodiments, the method further comprises longitudinally repeating steps (a) to (d) for the same transplant recipient and determining the longitudinal changes in the amount or a function thereof of RNA and the longitudinal changes in the amount or a function thereof of donor-derived RNA. In some embodiments, the method further comprises longitudinally repeating steps (a) to (d) for the same transplant recipient and determining the longitudinal changes in the amount or a function thereof of protein and the longitudinal changes in the amount or a function thereof of donor-derived protein.

[0200] Definitions

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] In some embodiments, for example, an allele refers to a nucleotide or nucleotide sequence that occupies a specific locus.

[0206] 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 "in" an individual. Genotype data can refer to output measurements from a genotyping platform that are made on genetic material.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] In some embodiments, for example, an allelic count refers to the number of sequences mapped to a specific locus, or if the locus is polymorphic, the number of sequences mapped to each of the alleles. If each allele is counted in a binary manner, the allelic count will be an integer. If a probabilistic count is made of the alleles, the allelic count can be a fraction.

[0212] 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 regions designed to hybridize to a targeted polymorphic locus and contains an initiation sequence designed to allow amplification such as by PCR. The primer can also contain a molecular barcode. The primer can contain a random region that is different for each individual molecule.

[0213] 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. These duplexes can then be physically separated from the sample in various ways.

[0214] 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 produce 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 was correctly determined.

[0215] In some embodiments, for example, mapping sequence reads is the process of determining the source location of a sequence read within the 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.

[0216] 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).

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] In some embodiments, for example, preferred enrichment of DNA or RNA corresponding to a locus, or preferred 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. Preferred enrichment can be performed on 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 preferred enrichment is performed on multiple loci, the enrichment level can refer to the average enrichment level of all loci in the locus group.

[0222] In some embodiments, for example, amplification refers to a technique for increasing the copy number of RNA and / or DNA molecules.

[0223] In some embodiments, for example, selective amplification can refer to a technique for increasing the copy number of specific RNA and / or DNA molecules or RNA and / or DNA molecules corresponding to a specific RNA and / or DNA region. Selective amplification can also refer to a technique for increasing the copy number of specific targeted RNA and / or DNA molecules or targeted RNA and / or DNA regions more than the copy number of non-targeted RNA and / or DNA molecules or regions. Selective amplification can be a method of preferred enrichment.

[0224] 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, 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 does not need to be related to the target sequence.

[0225] In some embodiments, for example, a universal adaptor or "ligation adaptor" 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 adaptor 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.

[0226] In some embodiments, for example, targeting refers to methods for selectively amplifying or otherwise preferentially enriching DNA or RNA molecules that correspond to a set of loci in a DNA or RNA mixture.

[0227] Analysis of donor-derived RNA for monitoring xenograft rejection

[0228] When the transplanted tissue is immunologically foreign, "acute rejection or AR" is the rejection by the immune system of the tissue transplant recipient. Acute rejection is characterized by infiltration of the transplanted tissue by the recipient's immune cells, which perform their effector functions and destroy 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.

[0229] "Chronic transplant rejection or injury" or "CAI" typically occurs in humans months to years after implantation, even in cases where immunosuppression of acute rejection is successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can generally be described by a set of specific conditions that are specific to a particular organ. For example, in lung transplantation, such conditions include airway fibroproliferative destruction (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, 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.

[0230] The term "xenograft rejection" encompasses both acute and chronic transplant rejection. The term xenotransplantation refers to a transplant in which the recipient and the donor are of different species. In particular, the recipient can be a human and the donor can be a pig. 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.

[0231] In some embodiments, the method includes performing universal amplification on the extracted RNA. In some embodiments, universal amplification preferentially amplifies donor-derived RNA rather than recipient-derived RNA.

[0232] In some embodiments, the transplant recipient is a mammal. In some embodiments, the transplant recipient is a human.

[0233] In some embodiments, the transplant recipient has received a transplant selected from organ transplant, tissue transplant, cell transplant, and fluid transplant. In some embodiments, the transplant recipient has received a transplant selected from the following: kidney transplant, liver transplant, pancreas transplant, intestine transplant, heart transplant, lung transplant, heart / lung transplant, stomach transplant, testis transplant, penis transplant, ovary transplant, uterus transplant, thymus transplant, face transplant, hand transplant, leg transplant, bone transplant, bone marrow transplant, corneal transplant, skin transplant, islet cell transplant, heart valve transplant, blood vessel transplant, and blood transfusion. In some embodiments, the transplant recipient has received a SPK transplant.

[0234] In some embodiments, the method further comprises detecting the occurrence or likely occurrence of active transplant rejection using a quantified amount of donor-derived RNA. In some embodiments, the method is performed without prior knowledge of the donor genotype.

[0235] 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.

[0236] In some embodiments, targeted amplification comprises amplifying at least 1,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 2,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 5,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying at least 10,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying from about 100 to about 50,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying from about 1,000 to about 50,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification comprises amplifying from about 5,000 to about 50,000 polymorphic loci in a single reaction volume.

[0237] 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.

[0238] 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.

[0239] In some embodiments, targeted amplification involves simultaneously amplifying 50 to 50,000 target loci in a single reaction volume using (i) at least 50 to 50,000 different primer pairs, or (ii) at least 50 to 50,000 target-specific primers and 50 to 50,000 universal or tag-specific primers.

[0240] In a further aspect, the present invention relates to a method for determining the likelihood of transplant rejection in a transplant recipient, the method comprising: extracting RNA from a blood sample of the transplant recipient, wherein the RNA comprises donor-derived RNA and recipient-derived RNA; performing universal amplification on the extracted RNA; performing targeted amplification at 50 to 50,000 target loci in a single reaction volume using 50 to 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 donor-derived RNA in the blood sample, wherein a larger amount of dd-RNA indicates a greater likelihood of transplant rejection.

[0241] In a further aspect, the present invention relates to a method for diagnosing that a xenotransplant in a xenotransplant recipient is undergoing acute rejection of the xenotransplant, the method comprising: extracting RNA from a blood sample of the xenotransplant recipient, wherein the RNA comprises donor-derived RNA and recipient-derived RNA; performing universal amplification on the extracted RNA; performing targeted amplification at 50 to 50,000 target loci in a single reaction volume using 50 to 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 donor-derived RNA in the blood sample, wherein an amount of dd-RNA greater than 1% (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 xenotransplant is undergoing acute rejection.

[0242] In some embodiments, xenotransplant rejection is antibody-mediated transplant rejection. In some embodiments, transplant rejection is T cell-mediated transplant rejection.

[0243] In some embodiments, an amount of dd-RNA less than 1% (or 0.9%, or 0.8%, or 0.7%, or 0.6%, or 0.5%) indicates that the xenotransplant is undergoing critical rejection, is undergoing other damage, or is stable.

[0244] In a further aspect, the present invention relates to a method for monitoring immunosuppressive therapy in a subject, the method comprising: extracting RNA from a blood sample of a xenograft recipient, wherein the RNA comprises donor-derived RNA and recipient-derived RNA; performing universal amplification on the extracted RNA; performing targeted amplification at 500 - 50,000 target loci in a single reaction volume using 500 - 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 donor-derived RNA in the blood sample, wherein a change in the dd-RNA level over a time interval indicates the xenograft status.

[0245] In some embodiments, the method further comprises adjusting the immunosuppressive therapy based on the dd-RNA level over a time interval.

[0246] In some embodiments, an increase in the dd-RNA level indicates transplant rejection and the need to adjust the immunosuppressive therapy. In some embodiments, no change or a decrease in the dd-RNA level indicates transplant tolerance or stability and the need to adjust the immunosuppressive therapy.

[0247] In some embodiments, an amount of dd-RNA greater than 1% (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 transplant is undergoing acute rejection. In some embodiments, the transplant rejection is antibody-mediated transplant rejection. In some embodiments, the transplant rejection is T cell-mediated transplant rejection.

[0248] In one aspect, the present disclosure relates to a method for administering immunosuppressive therapy in a xenograft recipient, the method comprising: (a) measuring the amount of cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (b) measuring the total amount of donor-derived cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dose of the immunosuppressive therapy based on the amount of cell-free DNA or a function thereof and the amount of donor-derived cell-free DNA or a function thereof.

[0249] In some embodiments, the amount of donor-derived cell-free DNA is measured by extracting cell-free DNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification of the extracted DNA at 200-50,000 target loci in a single reaction volume; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads.

[0250] In some embodiments, the methods herein further comprise longitudinally repeating steps (a) to (b) for the same transplant recipient, and determining the longitudinal changes in the amount of cell-free DNA or a function thereof and the longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

[0251] In some embodiments, the methods herein further comprise titrating the dose of the immunosuppressive therapy according to the longitudinal changes in the total amount of cell-free DNA or a function thereof and the longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

[0252] In one aspect, the present disclosure relates to a method of administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of RNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (b) measuring the amount of donor-derived RNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dose of the immunosuppressive therapy according to the amount of cell-free DNA or a function thereof and the amount of donor-derived RNA or a function thereof.

[0253] In some embodiments, the amount of donor-derived RNA is measured by extracting RNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; preparing an amplified cDNA composition by performing multiplex targeted amplification of complementary DNA (cDNA) derived from the extracted RNA at 200-50,000 animal target loci in a single reaction volume to detect and quantify the amount of animal donor-derived RNA; sequencing the amplified cDNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of donor-derived RNA based on the sequencing reads.

[0254] In some embodiments, the methods herein further comprise longitudinally repeating steps (a) to (b) for the same transplant recipient, and determining the longitudinal changes in the amount of RNA or a function thereof and the longitudinal changes in the amount of donor-derived RNA or a function thereof.

[0255] In some embodiments, the methods herein further include titrating the dosage of the immunosuppressive therapy based on longitudinal changes in the total amount of RNA or a function thereof and longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

[0256] In some embodiments, an amount of dd-RNA less than 1% (or 0.9%, or 0.8%, or 0.7%, or 0.6%, or 0.5%) indicates that the transplant is undergoing critical rejection, is undergoing other injury, or is stable.

[0257] In some embodiments, the method does not include genotyping the transplant donor and / or the transplant recipient.

[0258] In some embodiments, the method further includes measuring the amount of one or more alleles at a target locus that is a polymorphic locus.

[0259] In some embodiments, the xenograft recipient is human. In some embodiments, the xenograft recipient has received a xenograft selected from a kidney transplant, a liver transplant, a pancreas transplant, an islet cell transplant, an intestinal transplant, a heart transplant, a lung transplant, a bone marrow transplant, a heart valve transplant, or a skin transplant. In some embodiments, the xenograft recipient has received a SPK transplant.

[0260] In some embodiments, the extraction step includes size selection to enrich donor-derived RNA and reduce the amount of recipient-derived RNA lodged from burst blood cells.

[0261] In some embodiments, the universal amplification step preferentially amplifies donor-derived RNA rather than recipient-derived RNA lodged from burst blood cells.

[0262] In some embodiments, the method includes longitudinally collecting a plurality of blood samples from the xenograft recipient after transplantation and repeating steps (a) to (e) for each of the collected blood samples. In some embodiments, the method includes collecting and analyzing blood samples from the xenograft 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 includes collecting blood samples from the xenograft 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.

[0263] 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, where the cut-off threshold is 1% dd-RNA and the confidence interval is 95%.

[0264] 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-RNA and the confidence interval is 95%.

[0265] 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-RNA and the confidence interval is 95%.

[0266] 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-RNA and the confidence interval is 95%.

[0267] 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-RNA and the confidence interval is 95%.

[0268] 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-RNA and the confidence interval is 95%.

[0269] In some embodiments, the method has 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.

[0270] In some embodiments, the method has an accuracy determined by evaluating a linear value obtained from a linear regression analysis of measured donor fractions as a function of the corresponding attempted spike levels, where the linear value is an R2 value, and where the R2 value is from about 0.98 to about 1.0. In some embodiments, the R2 value is 0.999. In some embodiments, the method has an accuracy determined by calculating a slope value and an intercept value using a linear regression of the measured donor fractions as a function of the corresponding attempted spike levels, where the slope value is from about 0.9 to about 1.2, and the intercept value is from about -0.0001 to about 0.01. In some embodiments, the slope value is approximately 1, and the intercept value is approximately 0.

[0271] In some embodiments, the method has a precision determined by calculating a coefficient of variation (CV), where 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%.

[0272] In some embodiments, AR is antibody-mediated rejection (ABMR). In some embodiments, AR is T cell-mediated rejection (TCMR).

[0273] Also disclosed herein are methods for detecting xenotransplant donor-derived RNA (dd-RNA) in a sample from a xenotransplant recipient. In some embodiments, in the methods disclosed herein, the xenotransplant recipient is a mammal. In some embodiments, the xenotransplant recipient is a human. In some embodiments, the xenotransplant is from a pig, a primate, a baboon, a cow, or a dog.

[0274] In some embodiments, the transplant recipient has received a transplant selected from a kidney transplant, a liver transplant, a pancreas transplant, an islet cell transplant, an intestinal transplant, a heart transplant, a lung transplant, a bone marrow transplant, a heart valve transplant, or a skin transplant. In some embodiments, the transplant recipient has received a SPK transplant. In some embodiments, the method can be performed on the transplant recipient on the day of or after the transplant surgery, up to one year after the transplant surgery.

[0275] In some embodiments, disclosed herein is a method for amplifying a target locus of donor-derived RNA (dd-RNA) from a blood sample of a transplant recipient, the method comprising: a) extracting RNA from a blood sample of the transplant recipient, wherein the RNA comprises RNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted RNA at the target locus, wherein the target locus comprises 50 to 5000 target loci comprising polymorphic loci and non-polymorphic loci; and c) amplifying the target locus.

[0276] In some embodiments, a method for detecting donor-derived RNA (dd-RNA) in a blood sample from a transplant recipient is disclosed herein. The method includes: a) extracting RNA from a sample of the transplant recipient, wherein the RNA includes RNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted RNA at target loci, wherein the target loci include 50 to 5000 target loci including polymorphic loci and non-polymorphic loci; c) amplifying the target loci; d) contacting the amplified target loci with a probe that specifically hybridizes to the target loci; and e) detecting the binding of the target loci to the probe, thereby detecting the RNA in the blood sample. In some embodiments, the probe is labeled with a detectable marker.

[0277] In some embodiments, a method for determining the likelihood of transplant rejection in a transplant recipient is disclosed herein. The method includes: a) extracting RNA from a sample of the transplant recipient, wherein the RNA includes RNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted RNA at target loci, wherein the target loci include 50 to 5000 target loci including polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplanted RNA and the amount of recipient RNA in the recipient sample; wherein a greater amount of dd-RNA indicates a greater likelihood of transplant rejection.

[0278] In some embodiments, a method for diagnosing a transplant in a transplant recipient as undergoing acute rejection is disclosed herein. The method includes: a) extracting RNA from a blood sample of the transplant recipient, wherein the RNA includes RNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted RNA at target loci, wherein the target loci include 50 to 5000 target loci including polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplanted RNA and the amount of recipient RNA in the recipient sample; wherein an amount of dd-RNA greater than 1% (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 transplant is undergoing acute rejection.

[0279] In some embodiments, in the methods disclosed herein, the transplant rejection is antibody-mediated transplant rejection. In some embodiments, the transplant rejection is T cell-mediated transplant rejection. In some embodiments, an amount of dd-RNA less than 1% (or 0.9%, or 0.8%, or 0.7%, or 0.6%, or 0.5%) indicates that the transplant is undergoing borderline rejection, is undergoing other injury, or is stable.

[0280] In some embodiments, a method for monitoring an immunosuppressive therapy in a subject is disclosed herein, the method comprising: a) extracting RNA from a blood sample of a transplant recipient, wherein the RNA comprises RNA derived from both the transplanted cells and the xenotransplant recipient; b) enriching the extracted RNA at target loci, wherein the target loci comprise from 50 to 5000 target loci comprising polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of xenotransplanted DNA and the amount of recipient DNA in the recipient blood sample; wherein a change in the dd-RNA level over a time interval indicates the xenotransplant status. In some embodiments, the method further comprises adjusting the immunosuppressive therapy based on the dd-RNA level over the time interval. In some embodiments, an increase in the dd-RNA level indicates transplant rejection and the need to adjust the immunosuppressive therapy. In some embodiments, a change or decrease in the dd-RNA level indicates transplant tolerance or stability and the need to adjust the immunosuppressive therapy.

[0281] In some embodiments, in the methods disclosed herein, the target loci are amplified in amplicons having a length of about 50 bp to 100 bp, or a length of about 60 bp to 80 bp. In some embodiments, the amplicon has a length of about 65 bp.

[0282] In some embodiments, the methods disclosed herein further comprise measuring the amount of transplanted RNA and the amount of recipient RNA in the recipient blood sample.

[0283] In some embodiments, the methods disclosed herein do not include genotyping the xenotransplant donor and the xenotransplant recipient.

[0284] In some embodiments, the methods disclosed herein further comprise detecting the amplified target loci using a microarray.

[0285] In some embodiments, in the methods disclosed herein, the polymorphic loci and the non-polymorphic loci are amplified in a single reaction.

[0286] In some embodiments, in the methods disclosed herein, the RNA is preferably enriched at the target loci.

[0287] In some embodiments, preferentially enriching RNA in a sample at multiple target loci includes: obtaining a plurality of pre-circularized probes, where each probe targets one of the target loci, and where the 3' and 5' ends of the probes are designed to hybridize to an RNA region that is separated from the polymorphic site of the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 to 25, 26 to 30, 31 to 60, or a combination thereof; hybridizing the pre-circularized probes to RNA in the sample that has been converted to cDNA; filling the gap between the hybridized probe ends using a DNA polymerase; circularizing the pre-circularized probes; and amplifying the circularized probes.

[0288] In some embodiments, preferentially enriching RNA at multiple polymorphic loci includes: obtaining a plurality of ligation-mediated PCR probes, where each PCR probe targets one of the polymorphic loci, and where the upstream and downstream PCR probes are designed to hybridize to cDNA regions derived from the extracted RNA on one cDNA strand, the regions being separated from the polymorphic site of the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 to 25, 26 to 30, 31 to 60, or a combination thereof; hybridizing the ligation-mediated PCR probes to cDNA from a first sample; filling the gap between the ligation-mediated PCR probe ends using a DNA polymerase; ligating the ligation-mediated PCR probes; and amplifying the ligated ligation-mediated PCR probes.

[0289] In some embodiments, preferentially enriching RNA at multiple target loci includes: obtaining a plurality of hybridization capture probes that target specific loci; hybridizing the hybridization capture probes to RNA in the sample; and physically removing some or all of the unhybridized RNA from the first RNA sample.

[0290] 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 the 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.

[0291] In some embodiments, preferentially enriching RNA or RNA-derived cDNA at a plurality of polymorphic loci comprises: obtaining a plurality of internal forward primers, wherein each primer targets one of the polymorphic loci, and wherein the 3' end of the internal forward primer is designed to hybridize to a region of the RNA or RNA-derived cDNA that is upstream of the polymorphic site and separated from the polymorphic site 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, and wherein the 3' end of the internal reverse primer is designed to hybridize to a region of the RNA or RNA-derived cDNA that is upstream of the polymorphic site and separated from the polymorphic site 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 RNA or RNA-derived cDNA; and amplifying the cDNA using polymerase chain reaction to form amplicons.

[0292] In some embodiments, the method further comprises: 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 region of RNA or cDNA derived from RNA that is upstream of the internal forward primer; optionally 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 region of RNA or cDNA derived from RNA that is immediately downstream of the internal reverse primer; hybridizing a first primer to the RNA or cDNA derived from RNA; and amplifying the cDNA using polymerase chain reaction.

[0293] 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 region of RNA or cDNA derived from RNA that is 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 region of RNA or cDNA derived from RNA that is upstream of the internal forward primer; hybridizing a first primer to a region of the RNA or cDNA derived from RNA; and amplifying the RNA or cDNA derived from RNA using polymerase chain reaction.

[0294] In some embodiments, preparing the first sample further comprises attaching a universal adapter to the RNA in the first sample and amplifying the RNA in the first sample 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%.

[0295] In some embodiments, amplifying the RNA or cDNA derived from RNA is performed in one or more separate reaction volumes, and wherein each separate reaction volume contains 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.

[0296] In some embodiments, preparing the sample further comprises dividing the sample into multiple parts, and wherein the RNA or cDNA derived from the RNA in each part is preferentially enriched at a subset of a 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 universal primer sequences designed to allow PCR amplification. In some embodiments, at least some of the primers further contain random regions that are different for each individual primer molecule. In some embodiments, at least some of the primers further contain molecular barcodes.

[0297] In some embodiments, the method comprises: (a) in a single reaction volume, performing multiplex polymerase chain reaction (PCR) on a nucleic acid sample containing target loci using (i) at least 1,000 different primer pairs or (ii) at least 1,000 target-specific primers and universal or tag-specific primer pairs to simultaneously amplify at least 1,000 different target loci, thereby generating an amplification product containing target amplicons; and (b) sequencing the amplification product. In some embodiments, the method does not include using a microarray.

[0298] In some embodiments, the method comprises: (a) in a single reaction volume, performing multiplex polymerase chain reaction (PCR) on an RNA (or cDNA) sample containing target loci using (i) at least 1,000 different primer pairs or (ii) at least 1,000 target-specific primers and universal or tag-specific primer pairs to simultaneously amplify at least 1,000 different target loci, thereby generating an amplification product containing target amplicons; and b) sequencing the amplification product. In some embodiments, the method does not include using a microarray.

[0299] In some embodiments, the method further comprises obtaining genotype data from one or both of a transplant donor and a transplant recipient. In some embodiments, obtaining genotype data from one or both of a transplant donor and a transplant recipient comprises preparing RNA from the donor and recipient, wherein the preparation comprises preferentially enriching the RNA at a plurality of polymorphic loci to obtain prepared RNA or cDNA; optionally amplifying the prepared cDNA; and measuring the RNA at a plurality of polymorphic loci in the prepared sample.

[0300] In some embodiments, a joint distribution model of the expected allele count probabilities at multiple polymorphic loci on a chromosome is established using genetic data obtained from one or both of a transplant donor and a transplant recipient. In some embodiments, a first sample has been isolated from transplant recipient plasma, and wherein obtaining genotype data from the transplant recipient is accomplished by estimating recipient genotype data based on RNA measurements performed on the prepared sample.

[0301] In some embodiments, it is preferred that the enrichment results in the average degree of allelic bias between the prepared sample and the first sample having 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 multiple polymorphic loci are SNPs. In some embodiments, RNA in the prepared sample is measured by sequencing.

[0302] In some embodiments, a diagnostic kit for assisting in determining the transplant status of a transplant recipient is disclosed, wherein the diagnostic kit is capable of performing the preparation and measurement steps of the disclosed method.

[0303] In some embodiments, the allele count is probabilistic rather than binary. In some embodiments, measuring RNA in the prepared sample at multiple polymorphic loci is intended to determine whether the graft has inherited one or more linked haplotypes.

[0304] In some embodiments, a joint distribution model of allele count probabilities is established by using data on the probability of crossover at different positions of a chromosome within a chromosome to model the dependence between polymorphic alleles on the chromosome. In some embodiments, the steps of establishing the joint distribution model of allele counts and determining the relative probability of each hypothesis are accomplished using methods that do not require the use of a reference chromosome.

[0305] In some embodiments, determining the relative probability of each hypothesis utilizes the estimated fraction of donor-derived RNA (dd-RNA) in the prepared sample. In some embodiments, DNA measurements from the prepared sample for calculating allele count probabilities and determining the relative probability 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 transplant status corresponding to the hypothesis with the highest probability.

[0306] In some embodiments, determining the transplantation status 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, statistics only available when using donor genetic information, probabilities of normalized genotype signals for certain donor / receptor backgrounds, statistics calculated using the estimated transplantation fraction of the first sample or the prepared sample, and combinations thereof.

[0307] In some embodiments, a confidence estimate of the determined transplantation status is calculated. In some embodiments, the method further includes taking a clinical measure based on the determined transplantation status.

[0308] In some embodiments, the method disclosed herein is used to generate a report showing the determined transplantation status. In some embodiments, a kit for determining transplantation status 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 an RNA region immediately upstream and / or downstream of one of the target sites, wherein the hybridization region is separated from the target site 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.

[0309] In some embodiments, the method disclosed herein includes a selection step of selecting shorter RNAs.

[0310] In some embodiments, the method disclosed herein includes a general application step of enriching RNAs.

[0311] In some embodiments, determination that the amount of dd-RNA is above a cut-off threshold indicates acute rejection of the transplantation. Machine learning and artificial intelligence can be used to solve the problem of rejection versus non-rejection.

[0312] In some embodiments, the cut-off threshold is expressed as a percentage of dd-RNA in the blood sample (dd-RNA %).

[0313] In some embodiments, the cut-off threshold is expressed as the copy number of dd-RNA per unit volume of the blood sample.

[0314] In some embodiments, the cut-off threshold is expressed as the copy number of dd-RNA per unit volume of the blood sample multiplied by the body weight or blood volume of the transplant recipient.

[0315] In some embodiments, the cut-off threshold takes into account the patient's weight or blood volume.

[0316] In some embodiments, the cut-off threshold takes into account one or more of the following: donor genomic copies / plasma volume, cell-free DNA or RNA yield / plasma volume, donor height, donor weight, donor age, donor sex, donor race, donor organ mass, donor organ, living donor and deceased donor, related donor and 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), drugs (immunosuppressants, steroids, blood thinners, etc.), infections (BKV, EBV, CMV, UTI), recipient and / or donor HLA allele or epitope mismatches, Banff classification of renal allograft pathology, and cause and surveillance or protocol biopsies.

[0317] In some embodiments, the cut-off threshold is scaled according to the amount of total RNA in the blood sample.

[0318] In some embodiments, when the dd-RNA amount is higher than the cut-off threshold scaled according to the amount of total RNA in the sample and a 95% confidence interval, the method has at least 80% sensitivity in identifying acute rejection (AR) relative to non-AR.

[0319] In some embodiments, when the dd-RNA amount is higher than the cut-off threshold scaled according to the amount of total RNA 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.

[0320] In some embodiments, when the dd-RNA amount is higher than the cut-off threshold scaled according to the amount of total RNA in the 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-RNA amount is higher than the cut-off threshold scaled according to the amount of total RNA 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-RNA amount is higher than the cut-off threshold scaled according to the amount of total RNA 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-RNA amount is higher than the cut-off threshold scaled according to the amount of total RNA in the sample and a 95% confidence interval, the method has at least 95% sensitivity in identifying acute rejection (AR) relative to non-AR.

[0321] In some embodiments, when the amount of RNA is above a cut-off threshold scaled according to the amount of total RNA 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-RNA is above a cut-off threshold scaled according to the amount of total RNA 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-RNA is above a cut-off threshold scaled according to the amount of total RNA 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 above a cut-off threshold scaled according to the amount of total RNA 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-RNA is above a cut-off threshold scaled according to the amount of total RNA 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.

[0322] Analysis of donor-derived cell-free DNA for monitoring xenograft rejection

[0323] In one aspect, the invention further comprises: (i) measuring the amount of donor-derived cell-free DNA in a sample obtained from a xenograft recipient, extracting cell-free DNA from the sample obtained from the xenograft recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (ii) performing targeted amplification of the extracted DNA at 10 - 50,000 target loci in a single reaction volume; (iii) sequencing the amplified DNA to obtain sequencing reads and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads, determining transplant rejection based on whether the amount of donor-derived cell-free DNA or a function thereof exceeds a cut-off threshold indicating transplant rejection, wherein transplant rejection is determined based on whether the amount of donor-derived RNA and the amount of donor-derived cell-free DNA or a function thereof exceed a cut-off threshold indicating transplant rejection.

[0324] In another aspect, the present invention relates to a method for quantifying the amount of donor-derived cell-free DNA (dd-cfDNA) in a blood sample of a xenotransplant recipient, the method comprising: extracting DNA from a blood sample of a xenotransplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification at 100-50,000 or 500-50,000 target loci in a single reaction volume using 100-50,000 or 500-50,000 primer pairs, wherein the target loci comprise polymorphic loci and non-polymorphic loci, and wherein each primer pair is designed to amplify a target sequence of no more than 100 bp; and quantifying the amount of donor-derived cell-free DNA in the amplification product.

[0325] In another aspect, the present invention relates to a method for quantifying the amount of donor-derived cell-free DNA (dd-cfDNA) in a blood sample of a xenotransplant recipient, the method comprising: extracting DNA from a blood sample of a xenotransplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA, and wherein the extraction step comprises size selection to enrich donor-derived cell-free DNA and reduce the amount of recipient-derived cell-free DNA incorporated from lysed white blood cells; performing targeted amplification at 500-50,000 target loci in a single reaction volume using 500-50,000 primer pairs, wherein the target loci comprise polymorphic loci and non-polymorphic loci; and quantifying the amount of donor-derived cell-free DNA in the amplification product.

[0326] In another aspect, the present invention relates to a method for detecting donor-derived cell-free DNA (dd-cfDNA) in a blood sample of a transplant recipient, the method comprising: extracting DNA from a blood sample of a transplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free 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 product by high-throughput sequencing; and quantifying the amount of donor-derived cell-free DNA.

[0327] In some embodiments, the method further comprises performing universal amplification on the extracted DNA. In some embodiments, the universal amplification preferably amplifies donor-derived cell-free DNA rather than recipient-derived cell-free DNA incorporated from lysed white blood cells.

[0328] In some embodiments, the xenotransplant recipient is a mammal. In some embodiments, the transplant recipient is a human. In some embodiments, the xenotransplant is from a pig, a primate, a baboon, a cow, or a dog.

[0329] In some embodiments, the xenotransplant recipient has received a xenotransplant selected from an organ transplant, a tissue transplant, a cell transplant, and a fluid transplant. In some embodiments, the transplant recipient has received a transplant selected from the following: kidney transplant, liver transplant, pancreas transplant, intestine transplant, heart transplant, lung transplant, heart / lung transplant, stomach transplant, testicle transplant, penis transplant, ovary transplant, uterus transplant, thymus transplant, face transplant, hand transplant, leg transplant, bone transplant, bone marrow transplant, corneal transplant, skin transplant, islet cell transplant, heart valve transplant, blood vessel transplant, and blood transfusion. In some embodiments, the transplant recipient has received a SPK transplant.

[0330] In some embodiments, the quantification step includes determining the percentage of donor-derived cell-free DNA in the total amount of donor-derived cell-free DNA and recipient-derived cell-free DNA in the blood sample. In some embodiments, the quantification step includes determining the copy number of donor-derived cell-free DNA per unit volume of the blood sample.

[0331] In some embodiments, the method further includes detecting the occurrence or possible occurrence of active transplant rejection using the quantified amount of donor-derived cell-free DNA. In some embodiments, the method is performed without prior knowledge of the donor genotype.

[0332] 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.

[0333] In some embodiments, targeted amplification includes amplifying at least 1,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification includes amplifying at least 2,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification includes amplifying at least 5,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification includes amplifying at least 10,000 polymorphic loci in a single reaction volume. In some embodiments, targeted amplification includes amplifying 10 to 10,000, 10 to 50,000, 100 to 50,000, or 1,000 to 50,000 polymorphic loci in a single reaction volume.

[0334] 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, the polymorphic locus and the non-polymorphic locus are amplified in a single reaction.

[0335] In some embodiments, the quantification step comprises detecting the amplified target locus using a microarray. In some embodiments, the quantification step does not comprise using a microarray.

[0336] In some embodiments, targeted amplification comprises simultaneously amplifying 500 to 50,000 target loci in a single reaction volume using (i) at least 500 to 50,000 different primer pairs, or (ii) at least 500 to 50,000 target-specific primers and 500 to 50,000 universal or tag-specific primers.

[0337] In a further aspect, the present invention relates to a method for determining the likelihood of transplant rejection in a transplant recipient, the method comprising: extracting DNA from a blood sample of the transplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing universal amplification on the extracted DNA; performing targeted amplification at 500 to 50,000 target loci in a single reaction volume using 500 to 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 donor-derived cell-free DNA in the blood sample, wherein a larger amount of dd-cfDNA indicates a greater likelihood of transplant rejection.

[0338] In a further aspect, the present invention relates to a method for diagnosing a xenograft in a xenotransplant recipient as undergoing acute rejection, the method comprising: extracting DNA from a blood sample of the xenotransplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing universal amplification on the extracted DNA; performing targeted amplification at 500 to 50,000 target loci in a single reaction volume using 500 to 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 donor-derived cell-free DNA in the blood sample, wherein an amount of dd-cfDNA greater than 1% (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 xenograft is undergoing acute rejection.

[0339] In some embodiments, the xenograft rejection is antibody-mediated transplant rejection. In some embodiments, the xenograft rejection is T cell-mediated transplant rejection.

[0340] 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 xenograft is undergoing critical rejection, is undergoing other damage, or is stable.

[0341] In a further aspect, the present invention relates to a method of monitoring immunosuppressive therapy in a subject, the method comprising: extracting DNA from a blood sample of a transplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing universal amplification on the extracted DNA; performing targeted amplification at 500 - 50,000 target loci in a single reaction volume using 500 - 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 donor-derived cell-free DNA in the blood sample, wherein a change in the dd-cfDNA level over a time interval indicates the xenograft status.

[0342] In some embodiments, the method further comprises adjusting the immunosuppressive therapy based on the dd-cfDNA level over a time interval.

[0343] In some embodiments, an increase in the dd-cfDNA level indicates xenograft rejection and the need to adjust the immunosuppressive therapy. In some embodiments, no change or a decrease in the dd-cfDNA level indicates xenograft tolerance or stability and the need to adjust the immunosuppressive therapy.

[0344] In some embodiments, an amount of dd-cfDNA greater than 1% (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 xenograft is undergoing acute rejection. In some embodiments, the xenograft rejection is antibody-mediated transplant rejection. In some embodiments, the xenograft rejection is T cell-mediated transplant rejection.

[0345] 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 xenograft is undergoing critical rejection, is undergoing other damage, or is stable.

[0346] In some embodiments, the method does not include genotyping the transplant donor and / or the transplant recipient.

[0347] In some embodiments, the method further comprises measuring the amount of one or more alleles at a target locus that is a polymorphic locus.

[0348] In some embodiments, the target locus comprises 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.

[0349] In some embodiments, the target locus is amplified in an amplicon having a length of about 50 bp to 100 bp, or a length of about 50 bp to 90 bp, or a length of about 60 bp to 80 bp, or a length of about 60 bp to 75 bp, or a length of about 65 bp.

[0350] In some embodiments, the xenograft recipient is human. In some embodiments, the transplant recipient has received a transplant selected from a kidney transplant, a liver transplant, a pancreas transplant, an islet cell transplant, an intestinal transplant, a heart transplant, a lung transplant, a bone marrow transplant, a heart valve transplant, or a skin transplant. In some embodiments, the transplant recipient has received an SPK transplant.

[0351] In some embodiments, the extraction step comprises size selection to enrich donor-derived cell-free DNA and reduce the amount of recipient-derived cell-free DNA incorporated from lysed white blood cells.

[0352] In some embodiments, the universal amplification step preferably amplifies donor-derived cell-free DNA rather than recipient-derived cell-free DNA incorporated from lysed white blood cells.

[0353] In some embodiments, the method comprises longitudinally collecting a plurality of blood samples from the transplant recipient after transplantation and repeating steps (a) to (e) for each of the collected blood samples. 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.

[0354] 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, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0355] 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%.

[0356] 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 for identifying AR compared to non-AR, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0357] In some embodiments, the method has a sensitivity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% for identifying AR compared to a normal, stable allograft (STA), where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0358] In some embodiments, the method has a specificity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% for identifying AR compared to STA, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0359] 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 for identifying AR compared to STA, where the cut-off threshold is 1% dd-cfDNA and the confidence interval is 95%.

[0360] 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.

[0361] 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 the R 2 value is from about 0.98 to about 1.0. In some embodiments, the R 2The value is 0.999. In some embodiments, the method has an accuracy determined by calculating a slope value and an intercept value using linear regression of the measured donor fraction as a function of the corresponding attempted spike level, where the slope value is from about 0.9 to about 1.2 and the intercept value is from about -0.0001 to about 0.01. In some embodiments, the slope value is approximately 1 and the intercept value is approximately 0.

[0362] In some embodiments, the method has a precision determined by calculating the coefficient of variation (CV), where 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%.

[0363] In some embodiments, AR is antibody-mediated rejection (ABMR). In some embodiments, AR is T cell-mediated rejection (TCMR).

[0364] Also disclosed herein are methods for detecting donor-derived cell-free DNA (dd-cfDNA) from a sample of a xenotransplant recipient. In some embodiments, in the methods disclosed herein, the xenotransplant recipient is a mammal. In some embodiments, the xenotransplant recipient is a human. In some embodiments, the xenotransplant recipient has received a xenotransplant selected from kidney transplant, liver transplant, pancreas transplant, islet cell transplant, intestine transplant, heart transplant, lung transplant, bone marrow transplant, heart valve transplant, or skin transplant. In some embodiments, the xenotransplant recipient has received an SPK transplant. In some embodiments, the method can be performed on the xenotransplant recipient on or after the day of the transplant surgery until one year after the transplant surgery.

[0365] In some embodiments, disclosed herein is a method for amplifying a target locus of donor-derived cell-free DNA (dd-cfDNA) from a blood sample of a xenotransplant recipient, the method comprising: a) extracting DNA from a blood sample of the xenotransplant recipient, wherein the DNA comprises cell-free DNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted DNA at the target locus, wherein the target locus comprises 50 to 5000 target loci comprising polymorphic loci and non-polymorphic loci; and c) amplifying the target locus.

[0366] In some embodiments, a method for detecting donor-derived cell-free DNA (dd-cfDNA) in a blood sample from a transplant recipient is disclosed herein. The method includes: a) extracting DNA from a blood sample of the transplant recipient, wherein the DNA comprises cell-free DNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted DNA at target loci, wherein the target loci comprise 50 to 5000 target loci including polymorphic loci and non-polymorphic loci; c) amplifying the target loci; d) contacting the amplified target loci with a probe that specifically hybridizes to the target loci; and e) detecting the binding of the target loci to the probe, thereby detecting dd-cfDNA in the blood sample. In some embodiments, the probe is labeled with a detectable marker.

[0367] In some embodiments, a method for determining the likelihood of xenograft rejection in a transplant recipient is disclosed herein. The method includes: a) extracting DNA from a blood sample of the xenograft recipient, wherein the DNA comprises cell-free DNA derived from both the transplanted cells and the xenograft recipient; b) enriching the extracted DNA at target loci, wherein the target loci comprise 50 to 5000 target loci including polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of xenograft DNA and the amount of xenograft recipient DNA in the recipient's blood sample; wherein a relatively large amount of dd-cfDNA indicates a greater likelihood of xenograft rejection.

[0368] In some embodiments, a method for diagnosing a xenograft in a xenograft recipient as undergoing acute rejection is disclosed herein. The method includes: a) extracting DNA from a blood sample of the transplant recipient, wherein the DNA comprises cell-free DNA derived from both the transplanted cells and the xenograft recipient; b) enriching the extracted DNA at target loci, wherein the target loci comprise 50 to 5000 target loci including polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplanted DNA and the amount of recipient DNA in the recipient's blood sample; wherein an amount of dd-cfDNA greater than 1% (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 xenograft is undergoing acute rejection.

[0369] In some embodiments, in the methods disclosed herein, xenograft rejection is antibody-mediated transplant rejection. In some embodiments, xenograft rejection is T cell-mediated transplant rejection. 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 xenograft is undergoing critical rejection, is undergoing other damage, or is stable.

[0370] In some embodiments, disclosed herein is a method of monitoring an immunosuppressive therapy in a subject, the method comprising: a) extracting DNA from a blood sample of a transplant recipient, wherein the DNA comprises cell-free DNA derived from both the transplanted cells and the transplant recipient; b) enriching the extracted DNA at target loci, wherein the target loci comprise from 50 to 5000 target loci comprising polymorphic loci and non-polymorphic loci; c) amplifying the target loci; and d) measuring an amount of transplanted DNA and an amount of recipient DNA in the recipient blood sample; wherein a change in the dd-cfDNA level over a time interval indicates the xenograft status. In some embodiments, the method further comprises 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 xenograft rejection and a need to adjust the immunosuppressive therapy. In some embodiments, a change or decrease in the dd-cfDNA level indicates xenograft tolerance or stability and a need to adjust the immunosuppressive therapy.

[0371] In some embodiments, in the methods disclosed herein, the target loci are amplified in amplicons having a length of from about 50 bp to 100 bp, or having a length of from about 60 bp to 80 bp. In some embodiments, the amplicon has a length of about 65 bp.

[0372] Analysis method

[0373] In some embodiments, the method further comprises obtaining genotype data from one or both of the xenograft donor and the xenograft recipient. In some embodiments, obtaining genotype data from one or both of the xenograft donor and the xenograft recipient comprises preparing DNA from the donor and the recipient, wherein the preparation comprises 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 the plurality of polymorphic loci.

[0374] In some embodiments, a joint distribution model of the expected allele count probabilities at multiple polymorphic loci on a chromosome is established using genetic data obtained from one or both of a transplant donor and a transplant recipient. In some embodiments, a first sample has been isolated from transplant recipient plasma, and wherein obtaining genotype data from the transplant recipient is accomplished by estimating recipient genotype data based on DNA measurements performed on the prepared sample.

[0375] In some embodiments, enrichment is preferably such that the average degree of allelic 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 multiple polymorphic loci are SNPs. In some embodiments, DNA or RNA in the prepared sample is measured by sequencing.

[0376] In some embodiments, a diagnostic kit for assisting in determining the xenograft status of a xenograft recipient is disclosed, wherein the diagnostic kit is capable of performing the preparation and measurement steps of the disclosed method.

[0377] In some embodiments, the allele count is probabilistic rather than binary. In some embodiments, measuring DNA in the prepared sample at multiple polymorphic loci is also used to determine whether the xenograft has inherited one or more linked haplotypes.

[0378] In some embodiments, a joint distribution model of allele count probabilities is established by using data on the probability of crossover at different positions of a chromosome in a chromosome to model the dependence between polymorphic alleles on the chromosome. In some embodiments, the steps of establishing a joint distribution model of allele counts and determining the relative probability of each hypothesis are accomplished using methods that do not require the use of a reference chromosome.

[0379] In some embodiments, determining the relative probability of each hypothesis utilizes the estimated fraction of donor-derived RNA and / or donor-derived cell-free DNA (dd-cfDNA) in the prepared sample. In some embodiments, the DNA measurements from the prepared sample used to calculate allele count probabilities and determine the relative probability of each hypothesis include primary genetic data. In some embodiments, maximum likelihood estimation or maximum a posteriori estimation is used to perform the selection of the transplant status corresponding to the hypothesis with the highest probability.

[0380] In some embodiments, determining the xenograft status further includes combining the relative probabilities of each of the state hypotheses determined using the joint distribution model and allele count probabilities with the relative probabilities of each of the state hypotheses calculated using statistical techniques selected from the group consisting of: read count analysis, comparison of heterozygosity rates, statistics available only when using donor genetic information, probabilities of normalized genotype signals for certain donor / receptor backgrounds, statistics calculated using an estimated engraftment fraction of a first sample or a prepared sample, and combinations thereof.

[0381] In some embodiments, a confidence estimate of the determined xenograft status is calculated. In some embodiments, the method further includes taking a clinical measure based on the determined xenograft status.

[0382] In some embodiments, a report showing the determined xenograft status is generated using the method. In some embodiments, a kit for determining xenograft status 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, wherein each of the primers is designed to hybridize to a DNA region immediately upstream and / or downstream of one of the polymorphic sites located on the target chromosome, wherein the hybridization region is separated from the polymorphic site 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.

[0383] In some embodiments, the cut-off threshold takes into account one or more of the following: donor genome copies / plasma volume, cell-free DNA yield / plasma volume, donor height, donor weight, donor age, donor sex, donor race, donor organ mass, donor organ, living donor versus deceased donor, related donor versus 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), drugs (immunosuppressants, steroids, blood thinners, etc.), infections (BKV, EBV, CMV, UTI), recipient and / or donor HLA allele or epitope mismatches, Banff classification of renal allograft pathology, and cause versus surveillance or protocol biopsies.

[0384] In some embodiments, the cut-off threshold is scaled according to the amount of total cfDNA in the blood sample.

[0385] 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 80% sensitivity in identifying acute rejection (AR) relative to non-AR.

[0386] 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.

[0387] 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 80% sensitivity 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% sensitivity 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% sensitivity 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% sensitivity in identifying acute rejection (AR) relative to non-AR.

[0388] 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.

[0389] Multiplex amplification

[0390] In some embodiments, the method includes performing a multiplex amplification reaction before determining the sequences of the selectively enriched RNA or DNA to amplify multiple multi-target loci in one reaction mixture.

[0391] In certain illustrative embodiments, nucleic acid sequence data is generated by high-throughput RNA sequencing of multiple copies of a series of amplicons generated using a multiplex amplification reaction, wherein each amplicon of the series of amplicons spans at least one polymorphic locus in a set of polymorphic loci, and wherein each of the polymorphic loci in the set is amplified. In certain illustrative embodiments, nucleic acid sequence data is generated by high-throughput DNA sequencing of multiple copies of a series of amplicons generated using a multiplex amplification reaction, wherein each amplicon of the series of amplicons spans at least one polymorphic locus in a set of polymorphic loci, and wherein 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 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.

[0392] In some embodiments, amplification is performed using direct multiplex PCR, sequential PCR, nested PCR, double nested PCR, one-sided and semi-sided nested PCR, fully nested PCR, one-sided fully nested PCR, one-sided nested PCR, semi-nested PCR, semi-nested PCR, triple semi-nested PCR, semi-nested PCR, one-sided semi-nested PCR, reverse semi-nested PCR methods or one-sided PCR, as described in U.S. Application No. 13 / 683,604, filed Nov. 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed Nov. 18, 2011, U.S. Publication No. 2012 / 0270212, and U.S. Serial No. 61 / 994,791, filed May 16, 2014, all of which are hereby incorporated by reference in their entirety.

[0393] In some embodiments, multiplex PCR is used. In some embodiments, a method of amplifying target loci in a nucleic acid sample involves (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 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 comprising 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 not immobilized on a solid support. In some embodiments, the primers are not part of a microarray.

[0394] In certain embodiments, at least 1 / 2 of the multiplex amplification reactions are carried out 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.

[0395] In certain embodiments, the multiplex amplification reaction can comprise, for example, between 2,500 and 50,000 multiplex reactions. In certain embodiments, multiplex reactions in the following ranges are carried out: with the lower end of the range being between 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 being between 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, 50,000, and 100,000.

[0396] In one embodiment, multiplex PCR assays are 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 50 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 (50-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 a blood, plasma, serum, solid tissue, or urine sample. 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 parallel 5,000-plex assays are performed. In another embodiment, the original cfDNA sample is divided into n samples, and parallel (about 10,000 / n)-plex assays are performed, where n is between 2 and 12, or between 12 and 24, or between 24 and 48, or between 48 and 96.

[0397] In one embodiment, the methods disclosed herein use highly 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 with each other. PCR probes, commonly referred to as primers, are selected by creating a thermodynamic model of 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 this model to eliminate designs that are incompatible with other designs in the pool. Another technique that allows highly multiplexed targeted PCR to be performed 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 for multiplexing of 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 for 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.

[0398] Bioinformatics methods are used to analyze the genetic data obtained from multiplex PCR. Bioinformatics methods useful and relevant to the methods disclosed herein can be found in U.S. Patent Publication No. 2018 / 0025109, which is incorporated herein by reference.

[0399] High-throughput sequencing

[0400] In some embodiments, the sequence of the amplicon is determined by performing high-throughput sequencing.

[0401] 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, ILLUMINA's 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 of these methods physically transform the genetic data stored in the DNA sample into a set of genetic data, which is typically stored in a memory device for processing.

[0402] 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.

[0403] 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 a specific time (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) because 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, that is, the number of droplets that fluoresce due to correct amplification. This positive fraction accurately indicates the initial amount of the template nucleic acid.

[0404] Tracer DNA and its uses

[0405] A tracer DNA for estimating the amount of total cfDNA in a sample is described in U.S. Provisional Application No. 63 / 031,879, filed on May 29, 2020, entitled "Improved Methods for Detection of Donor Derived Cell-Free DNA", which is incorporated herein by reference in its entirety. In some embodiments, the tracer DNA comprises a synthetic double-stranded DNA molecule. In some embodiments, the tracer DNA comprises a DNA molecule of non-human origin.

[0406] In some embodiments, the tracer DNA comprises a DNA molecule having a length of about 50 bp to 500 bp, or about 75 bp to 300 bp, or about 100 bp to 250 bp, or about 125 bp to 200 bp, or about 125 bp, or about 160 bp, or about 200 bp or about 500 bp to 1,000 bp.

[0407] In some embodiments, the tracer DNA comprises DNA molecules having the same or substantially the same length, such as DNA molecules having a length of about 125 bp, or about 160 bp or about 200 bp. In some embodiments, the tracer DNA comprises DNA molecules having different lengths, such as a first DNA molecule having a length of about 125 bp, a second DNA molecule having a length of about 160 bp, and a third DNA molecule having a length of about 200 bp. In some embodiments, DNA molecules having different lengths are used to determine the size distribution of the cell-free DNA in the sample.

[0408] In some embodiments, the tracer DNA comprises a target sequence, wherein the target sequence comprises a barcode located between a pair of primer binding sites capable of binding to a pair of primer pairs. In some embodiments, at least a portion of the tracer DNA is designed based on an endogenous human SNP locus by replacing the endogenous sequence containing the SNP locus with a barcode. In the mmPCR target enrichment step, the primer pair targeting the SNP locus can also amplify the portion of the tracer DNA containing the barcode.

[0409] In some embodiments, the barcode is an arbitrary barcode. In some embodiments, the barcode comprises the reverse complement of the corresponding endogenous genomic sequence that can be amplified by the same primer pair.

[0410] In some embodiments, one or both sides of the target sequence within the tracer DNA are flanked by endogenous genomic sequences. In some embodiments, one or both sides of the target sequence within the tracer DNA are flanked by non-endogenous sequences.

[0411] In some embodiments, the tracer DNA includes a plurality of target sequences. In some embodiments, the tracer DNA includes a first target sequence, the first target sequence including a first barcode positioned between a first pair of primer binding sites capable of binding to a first pair of primers, and a second barcode positioned between a second pair of primer binding sites capable of binding to a second pair of primers. In some embodiments, the first and / or second target sequences are designed based on one or more endogenous human SNP loci by replacing the endogenous sequence containing the SNP locus with a barcode. In some embodiments, the first and / or second barcodes are arbitrary barcodes. In some embodiments, the first and / or second barcodes include the reverse complement of the corresponding endogenous genomic sequence that can be amplified by the first or second primer pair. In some embodiments, one or both sides of the first and / or second target sequences within the tracer DNA are flanked by endogenous genomic sequences. In some embodiments, one or both sides of the first and / or second target sequences within the tracer DNA are flanked by non-endogenous sequences.

[0412] In some embodiments, the tracer DNA comprises DNA molecules having the same or substantially the same sequence. In some embodiments, the tracer DNA includes DNA molecules having different sequences.

[0413] In some embodiments, the tracer DNA includes a first DNA, the first DNA including a first target sequence, and a second DNA, the second DNA including a second target sequence. In some embodiments, the first target sequence and the second target sequence have different barcodes positioned between the same primer binding sites. In some embodiments, the first target sequence and the second target sequence have different barcodes positioned between the same primer binding sites, wherein the different barcodes have the same or substantially the same length. In some embodiments, the first target sequence and the second target sequence have different barcodes positioned between the same primer binding sites, wherein the different barcodes have different lengths. In some embodiments, the first target sequence and the second target sequence are designed based on different endogenous human SNP loci and thus include different primer binding sites. In some embodiments, the amount of the first DNA and the amount of the second DNA in the tracer DNA are the same or substantially the same. In some embodiments, the amount of the first DNA and the amount of the second DNA in the tracer DNA are different.

[0414] In certain embodiments, the tracer DNA can be used to improve the accuracy and precision of the methods described herein, facilitate quantification over a greater input range, evaluate the efficiency of different steps over different size ranges, and / or calculate the fragment size distribution of the input material.

[0415] Some embodiments of the present invention relate to a method for quantifying the amount of total cell-free DNA in a biological sample, the method comprising: a) isolating cell-free DNA from a biological sample, wherein a first tracer DNA is added before or after the isolation of cell-free DNA; b) performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs; c) sequencing the amplification products by high-throughput sequencing to generate sequencing reads; and d) using the sequencing reads derived from the first tracer DNA to quantify the amount of total cell-free DNA.

[0416] In some embodiments, the method comprises adding the first tracer DNA to a whole blood sample before plasma extraction. In some embodiments, the method comprises adding the first tracer DNA to a plasma sample after plasma extraction and before isolating the cell-free DNA. In some embodiments, the method comprises adding the first tracer DNA to a composition comprising the isolated cell-free DNA. In some embodiments, the method comprises ligating an adaptor to the isolated cell-free DNA to obtain a composition comprising adaptor-ligated DNA, and adding the first tracer DNA to the composition comprising adaptor-ligated DNA.

[0417] In some embodiments, the method further comprises adding a second tracer DNA before the targeted amplification. In some embodiments, the method further comprises adding a second tracer DNA after the targeted amplification.

[0418] In some embodiments, the amount of total cfDNA in a sample is estimated using the NOR (identifiable by barcode) of the tracer DNA, the NOR of the sample DNA, and the known amount of tracer DNA added to the plasma sample. In some embodiments, the ratio between the NOR of the tracer DNA and the NOR of the sample DNA is used to quantify the amount of total cell-free DNA. In some embodiments, the ratio between the NOR of the barcode and the NOR of the corresponding endogenous genomic sequence is used to quantify the amount of total cell-free DNA. In some embodiments, this information, together with the plasma volume, can also be used to calculate the amount of cfDNA per volume of plasma. In some embodiments, these can be multiplied by the percentage of donor DNA to calculate the total amount of donor cfDNA and the donor cfDNA per volume of plasma.

[0419] Accordingly, in another aspect, the present invention relates to a method for quantifying the amount of total cell-free DNA in a biological sample, the method comprising: a) isolating cell-free DNA from the biological sample, wherein a first tracer DNA composition is added before or after the isolation of the cell-free DNA; b) performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs; c) sequencing the amplification products by high-throughput sequencing to generate sequencing reads; and d) quantifying the amount of total cell-free DNA using the sequencing reads derived from the first tracer DNA composition.

[0420] In a further aspect, the present invention relates to a method for quantifying the amount of donor-derived cell-free DNA in a biological sample of a transplant recipient, the method comprising: a) isolating cell-free DNA from the biological sample of the transplant recipient, wherein the isolated cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA, and wherein a first tracer DNA composition is added before or after the isolation of the cell-free DNA; b) performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs; c) sequencing the amplification products by high-throughput sequencing to generate sequencing reads; and d) quantifying the amount of donor-derived cell-free DNA and the amount of total cell-free DNA, wherein the amount of total cell-free DNA is quantified using the sequencing reads derived from the first tracer DNA composition.

[0421] In a further aspect, the present invention relates to a method for determining the occurrence or possible occurrence of transplant rejection, the method comprising: a) isolating cell-free DNA from the biological sample of the transplant recipient, wherein the isolated cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA, and wherein a first tracer DNA composition is added before or after the isolation of the cell-free DNA; b) performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs; c) sequencing the amplification products by high-throughput sequencing to generate sequencing reads; d) quantifying the amount of donor-derived cell-free DNA and the amount of total cell-free DNA, wherein the amount of total cell-free DNA is quantified using the sequencing reads derived from the first tracer DNA composition, and using the amount of donor-derived cell-free DNA, determining the occurrence or possible occurrence of transplant rejection by comparing the amount of donor-derived cell-free DNA with a threshold value, wherein the threshold value is determined based on the amount of total cell-free DNA.

[0422] In some embodiments, the threshold value is a function of the number of sequencing reads of the donor-derived cell-free DNA.

[0423] In some embodiments, the method further comprises labeling the sample if the amount of total cell-free DNA exceeds a predetermined range. In some embodiments, the method further comprises labeling the sample if the amount of total cell-free DNA is higher than a predetermined value. In some embodiments, the method further comprises labeling the sample if the amount of total cell-free DNA is lower than a predetermined value.

[0424] In some embodiments, the method comprises adding the first tracer DNA composition to a whole blood sample prior to plasma extraction. In some embodiments, the method comprises adding the first tracer DNA composition to a plasma sample after plasma extraction and prior to separating the cell-free DNA. In some embodiments, the method comprises adding the first tracer DNA composition to a composition comprising the separated cell-free DNA. In some embodiments, the method comprises ligating an adaptor to the separated cell-free DNA to obtain a composition comprising adaptor-ligated DNA, and adding the first tracer DNA composition to the composition comprising adaptor-ligated DNA.

[0425] In some embodiments, the method further comprises adding a second tracer DNA composition prior to the targeted amplification. In some embodiments, the method further comprises adding a second tracer DNA composition after the targeted amplification.

[0426] In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition comprises a plurality of DNA molecules having different sequences.

[0427] In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition comprises a plurality of DNA molecules having different concentrations.

[0428] In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition comprises a plurality of DNA molecules having different lengths. In some embodiments, a plurality of DNA molecules having different lengths are used to determine the size distribution of the cell-free DNA in the sample.

[0429] In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition comprises a plurality of DNA molecules of non-human origin.

[0430] In some embodiments, each of the first tracer DNA composition and / or the second tracer DNA composition includes a target sequence, wherein the target sequence includes a barcode positioned between a pair of primer binding sites capable of binding to a pair of primer pairs. In some embodiments, the barcode includes the reverse complement of a corresponding endogenous genomic sequence that can be amplified by the same primer pair.

[0431] In some embodiments, the ratio between the read count of the tracer DNA and the read count of the sample DNA is used to quantify the amount of total cell-free DNA. In some embodiments, the ratio between the read count of the barcode and the read count of the corresponding endogenous genomic sequence is used to quantify the amount of total cell-free DNA.

[0432] In some embodiments, one or both sides of the target sequence are flanked by endogenous genomic sequences. In some embodiments, one or both sides of the target sequence are flanked by non-endogenous sequences.

[0433] In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a synthetic double-stranded DNA molecule. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of 50 bp to 500 bp. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of 75 bp to 300 bp. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of 100 bp to 250 bp. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of 125 bp to 200 bp. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of approximately 200 bp. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of approximately 160 bp. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of approximately 125 bp. In some embodiments, the first tracer DNA composition and / or the second tracer DNA composition includes a DNA molecule having a length of 500 bp to 1,000 bp.

[0434] In some embodiments, the targeted amplification includes amplifying at least 100 polymorphic or SNP loci in a single reaction volume. In some embodiments, the targeted amplification includes amplifying at least 200 polymorphic or SNP loci in a single reaction volume. In some embodiments, the targeted amplification includes amplifying at least 500 polymorphic or SNP loci in a single reaction volume. In some embodiments, the targeted amplification includes amplifying at least 1,000 polymorphic or SNP loci in a single reaction volume. In some embodiments, the targeted amplification includes amplifying at least 2,000 polymorphic or SNP loci in a single reaction volume. In some embodiments, the targeted amplification includes amplifying at least 5,000 polymorphic or SNP loci in a single reaction volume. In some embodiments, the targeted amplification includes amplifying at least 10,000 polymorphic or SNP loci in a single reaction volume.

[0435] In some embodiments, each primer pair is designed to amplify a target sequence of about 35 bp to 200 bp. 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 about 60 bp to 75 bp. In some embodiments, each primer pair is designed to amplify a target sequence of about 65 bp.

[0436] In some embodiments, the transplant recipient is a human subject. In some embodiments, the transplant recipient has received an allograft. In some embodiments, the transplant recipient has received a xenograft.

[0437] In some embodiments, the transplantation is a human transplantation. In some embodiments, the transplantation is a porcine transplantation. In some embodiments, the transplantation is from a non-human animal.

[0438] In some embodiments, the transplantation is an organ transplantation, a tissue transplantation, or a cell transplantation. In some embodiments, the transplantation is a kidney transplantation, a liver transplantation, a pancreas transplantation, an intestine transplantation, a heart transplantation, a lung transplantation, a heart / lung transplantation, a stomach transplantation, a testis transplantation, a penis transplantation, an ovary transplantation, a uterus transplantation, a thymus transplantation, a face transplantation, a hand transplantation, a leg transplantation, a bone transplantation, a bone marrow transplantation, a corneal transplantation, a skin transplantation, an islet cell transplantation, a heart valve transplantation, a blood vessel transplantation, or a blood transfusion.

[0439] In some embodiments, the method further includes determining the transplant rejection as antibody-mediated transplant rejection, T cell-mediated transplant rejection, transplant injury, viral infection, bacterial infection, or borderline rejection.

[0440] Working Examples

[0441] Example 1

[0442] This example is illustrative only, and one of ordinary skill in the art will understand that the invention disclosed herein can be implemented in a variety of other ways.

[0443] Blood sample

[0444] Adult male and female or young adult patients receive donor organs from related or unrelated living donors or unrelated deceased donors. The time points for blood sampling in patients after transplantation surgery are at the time of allograft biopsy or at various pre-specified time intervals based on laboratory protocols. Generally, the samples are biopsy-matched and blood is drawn at the time of clinical dysfunction and biopsy or at protocol biopsy (when most patients have no clinical dysfunction). Additionally, some patients have serial blood draws after transplantation. The selection of study samples is based on (a) having sufficient plasma available and (b) whether the sample is associated with biopsy information. In the entire cohort of 300 samples, 72.3% were drawn on the day of biopsy.

[0445] Nucleic acid measurement in blood samples

[0446] Using QIAAMP TM Circulating Nucleic Acid Kit (Qiagen) to extract nucleic acids, such as RNA or DNA, especially cell-free DNA, mRNA, and microRNA, from plasma samples, and using LABCHIP TM NGS 5k Kit (Perkin Elmer, Waltham, MA, USA) for quantification. Library preparation was performed using the Natera Library Prep Kit as described in Abbosh et al., Nature 545:446 - 451 (2017), and the 18 cycles of library amplification were modified to stabilize the library. Using LABCHIP TM NGS 5k to quantify the purified library as described in Abbosh et al., Nature 545:446 - 451 (2017). Target enrichment was accomplished using large-scale multiplex PCR (mmPCR), which uses a modified version described in Zimmermann et al., Prenat. Diagn. 32:1233 - 1241 (2012), where 13,392 single nucleotide polymorphisms (SNPs) were targeted. Then, the amplicons were sequenced on an Illumina HiSeq 2500 for 50 cycles single-end, with 10 to 11 million reads per sample.

[0447] Statistical analysis of nucleic acids, dd-cfDNA, and eGFR

[0448] In each sample, donor-derived RNA and / or dd-cfDNA was measured and correlated with the rejection status, and the results were compared with eGFR. All statistical tests were two-sided where applicable. Significance was set at p < 0.05. Since the distribution of patients' dd-cfDNA was severely skewed across groups, the Kruskal-Wallis rank sum test was used, followed by the Dunn multiple comparison test with Holm correction to analyze the data. eGFR (serum creatinine in mg / dL) was calculated as previously described and was used for adult and pediatric patients. Briefly, eGFR = 186 × serum creatinine -1.154 × age -0.203 × (1.210 if black) × (0.742 if female).

[0449] Cas9 / Cas12a or Cas13 CRISPR technology was used to remove contamination. The Cas9 / Cas12a CRISPR system can be used to remove contaminants from cDNA libraries. The Cas13 system can be used to remove contaminating RNA from biological samples.

[0450] Example 2

[0451] This example evaluated the xenograft monitoring test as disclosed herein. Figure 1 An overview of a test method for detecting cell-free DNA from an animal donor in a human plasma sample is shown.

[0452] First, cell-free DNA (cfDNA) was purified from human and porcine plasma samples. Next, a combination of porcine and human cell-free DNA was made. This combination was tested for mixtures containing porcine cell-free DNA from 100% down to 0.01%. A sequencing library was prepared from the combination of porcine and human cfDNA.

[0453] The porcine / human cfDNA ratio of each combination was determined by whole-genome sequencing of the prepared library, and the determined porcine / human cfDNA ratio was plotted against the actual ratio for each sample, as Figure 2 shown. Linear regression analysis showed that there was a linear relationship (R 2 = 0.9961) between the measured and actual porcine / human cfDNA ratios. Thus, this evaluation demonstrated the concept of using whole-genome sequencing of cfDNA extracted from plasma samples to measure the amount of cfDNA from xenografts, which is useful for monitoring xenograft rejection.

[0454] ****

Claims

1. A method for preparing a non-naturally occurring DNA composition, the DNA being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) Extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) Preparing a sequencing library from the extracted cell-free DNA, and sequencing the sequencing library by high-throughput sequencing to obtain sequencing reads, and quantifying the total amount of donor-derived cell-free DNA based on the sequencing reads; (c) Determining whether the amount of donor-derived cell-free DNA from the xenograft or a function thereof exceeds a cut-off threshold indicative of xenograft rejection or injury.

2. A method for preparing a non-naturally occurring amplified DNA composition, the DNA being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) Extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA from the xenograft and recipient-derived cell-free DNA; (b) Preparing an amplified DNA composition by multiplex targeted amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume to detect and quantify the amount of donor-derived cell-free DNA, wherein the target loci comprise a set of animal target loci and a set of human target loci; (c) Determining whether the amount of donor-derived cell-free DNA or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

3. A method for preparing a non-naturally occurring amplified DNA composition, the DNA being derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method for determining xenograft rejection, the method comprising: (a) Extracting cell-free DNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) Preparing an amplified DNA composition by targeted amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise both human target loci and animal target loci, and sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amounts of both donor-derived cell-free DNA and recipient-derived cell-free DNA from the xenotransplant based on the sequencing reads, wherein the human loci and the animal loci are the same, and wherein the human reads and the animal reads are distinguished based on the inserted sequences; (c) Determining whether the fraction of donor-derived cell-free DNA from the animal or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

4. A method for preparing a non-naturally occurring amplified DNA composition, wherein the DNA is derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenotransplant rejection, the method comprising: (a) extracting cell-free DNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing an amplified DNA composition by targeting amplification of the extracted cell-free DNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise one or more target loci indicative of xenotransplant rejection; and (c) determining the amount of the one or more target loci indicative of xenotransplant rejection, and determining whether the amount of the one or more target loci indicative of xenotransplant rejection or a function thereof exceeds a cut-off threshold indicative of xenotransplant rejection.

5. The method according to any one of claims 1 to 4, wherein the DNA composition comprises one or more target loci indicative of xenotransplant rejection; and wherein the determining step further comprises determining the amount of the one or more target loci indicative of xenotransplant rejection, and determining whether the amount of the one or more target loci indicative of xenotransplant rejection or a function thereof exceeds a cut-off threshold indicative of xenotransplant rejection; wherein xenotransplant rejection is determined by a combination of (i) the amount of the one or more target loci indicative of xenotransplant rejection or a function thereof and (ii) the total amount or fraction of animal donor-derived cell-free DNA.

6. A method for preparing a non-naturally occurring complementary DNA (cDNA) composition from RNA, wherein the RNA is extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenotransplant rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; (b) preparing a cDNA sequencing library from the extracted RNA, sequencing the cDNA sequencing library by high-throughput sequencing to obtain sequencing reads, and quantifying the total amount of donor-derived RNA based on the sequencing reads; (c) determining whether the total amount of donor-derived RNA from the xenotransplant or a function thereof exceeds a cut-off threshold indicative of xenotransplant rejection.

7. A method for preparing a non-naturally occurring amplified complementary DNA (cDNA) composition from RNA, wherein the RNA is extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, and the method is for determining xenotransplant rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA from the xenotransplant and recipient-derived RNA; (b) Preparing an amplified cDNA composition by multiplex targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume to detect and quantify the amount of donor-derived RNA, wherein the target loci comprise an animal target locus set and a human target locus set; (c) Determining whether the amount of donor-derived RNA target loci or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

8. A method for preparing an unnaturally occurring amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient for determining xenograft rejection, the method comprising: (a) Extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; (b) Preparing an amplified cDNA composition by targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise both human target loci and animal target loci, and sequencing the amplified cDNA by high-throughput sequencing to obtain sequence reads, and quantifying the amounts of both donor-derived RNA and xenograft recipient-derived RNA based on the sequence reads, wherein the human loci and the animal loci are the same, and wherein human reads and animal reads are distinguished based on the inserted sequences; (c) Determining whether the fraction of donor-derived RNA or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

9. A method for preparing an unnaturally occurring amplified complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient for determining xenograft rejection, the method comprising: (a) Extracting RNA from the blood, plasma, serum, or urine sample of the xenograft recipient, wherein the extracted RNA comprises donor-derived cell-free RNA and recipient-derived RNA; (b) Preparing an amplified cDNA composition by targeted amplification of cDNA derived from the extracted RNA at 10 - 50,000 target loci in a single reaction volume, wherein the target loci comprise one or more target loci indicative of xenograft rejection; and (c) Determining the amount of the one or more target loci indicative of xenograft rejection, and whether the amount of the one or more target loci indicative of xenograft rejection or a function thereof exceeds a cut-off threshold indicative of xenograft rejection.

10. The method according to any one of claims 6 to 9, wherein the composition comprises target loci indicative of xenograft rejection; and wherein the determining step further comprises determining the amount of the one or more target loci indicative of xenograft rejection, and determining whether the amount or a function thereof of the one or more target loci indicative of xenograft rejection exceeds a cut-off threshold indicative of xenograft rejection, wherein xenograft rejection is determined by a combination of the amount or a function thereof of the one or more target loci indicative of xenograft rejection and the total amount of RNA from an animal donor source or a fraction of RNA from an animal donor source.

11. A method for preparing a non-naturally occurring protein composition, the protein being derived from a blood, plasma, serum or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting protein from the blood, plasma, serum or urine sample of the xenograft recipient, wherein the protein comprises donor-derived protein and recipient-derived protein from the xenograft; (b) detecting and quantifying the amount of the donor-derived protein; (c) determining whether the amount or a function thereof of the donor-derived protein exceeds a cut-off threshold indicative of xenograft rejection.

12. A method for preparing a non-naturally occurring protein composition, the protein being derived from a blood, plasma, serum or urine sample of a xenograft recipient, the method for determining xenograft rejection, the method comprising: (a) extracting protein from the blood, plasma, serum or urine sample of the xenograft recipient, wherein the protein comprises donor-derived protein and recipient-derived protein from the xenograft; (b) detecting and quantifying the amount of one or more target proteins, wherein the one or more target proteins comprise both recipient-derived protein and donor-derived protein, and wherein the one or more target proteins are indicative of xenograft rejection; (c) determining whether the amount or a function thereof of the one or more target proteins exceeds a cut-off threshold indicative of xenograft rejection.

13. A method for administering an immunosuppressive therapy to a xenograft recipient, the method comprising: (a) measuring the amount of donor-derived protein according to claim 11; and (b) titrating the dose of the immunosuppressive therapy according to the amount or a function thereof of the donor-derived protein.

14. A method for administering an immunosuppressive therapy to a xenograft recipient, the method comprising: (a) measuring the amount of one or more target proteins according to claim 12; and (b) titrating the dose of the immunosuppressive therapy according to the amount or a function thereof of the one or more target proteins.

15. The method according to any one of claims 13 to 14, further comprising longitudinally repeating steps (a) to (b) on the same xenograft recipient, and determining the longitudinal changes in the amount or a function thereof of the donor-derived protein, the donor-derived target protein, and the amount or a function thereof of the donor-derived protein, the target protein.

16. The method according to claim 15, further comprising titrating the dose of the immunosuppressive therapy according to longitudinal changes in the protein from the donor source, the donor-derived target protein, or a function thereof.

17. The method according to any one of claims 11 to 16, wherein the protein is derived from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenotransplant recipient, and the method comprises extracting the protein from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenotransplant recipient.

18. A method of administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (b) measuring the total amount of donor-derived cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dose of the immunosuppressive therapy according to the amount of cell-free DNA or a function thereof and the amount of donor-derived cell-free DNA or a function thereof.

19. The method according to claim 18, wherein the amount of donor-derived cell-free DNA is measured by: extracting cell-free DNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; targeted amplification of the extracted DNA at 200 - 50,000 target loci in a single reaction volume; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads.

20. The method according to any one of claims 18 to 19, further comprising longitudinally repeating steps (a) to (b) for the same transplant recipient, and determining longitudinal changes in the amount of cell-free DNA or a function thereof and longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

21. The method according to claim 20, further comprising titrating the dose of the immunosuppressive therapy according to longitudinal changes in the total amount of cell-free DNA or a function thereof and longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

22. A method of administering an immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of RNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (b) measuring the amount of donor-derived RNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dose of the immunosuppressive therapy according to the amount of cell-free DNA or a function thereof and the amount of donor-derived RNA or a function thereof.

23. The method according to claim 20, wherein the amount of donor-derived RNA is measured by: extracting RNA from the blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; An amplified cDNA composition is prepared by multiplex targeted amplification of complementary DNA (cDNA) derived from the extracted RNA at 200 - 50,000 animal target loci in a single reaction volume to detect and quantify the amount of RNA from an animal donor; The amplified cDNA is sequenced by high-throughput sequencing to obtain sequencing reads, and the amount of donor-derived RNA is quantified based on the sequencing reads.

24. The method according to any one of claims 22 to 23, further comprising longitudinally repeating steps (a) to (b) for the same transplant recipient, and determining the longitudinal changes in the amount of RNA or a function thereof and the longitudinal changes in the amount of donor-derived RNA or a function thereof.

25. The method according to claim 24, further comprising titrating the dose of the immunosuppressive therapy according to the longitudinal changes in the total amount of RNA or a function thereof and the longitudinal changes in the amount of donor-derived cell-free DNA or a function thereof.

26. The method according to any one of claims 1 to 10 and 18 to 21, wherein an internal control is added to the sample.

27. The method according to any one of claims 1 to 10 and 18 to 21, wherein the sequencing includes shotgun whole-genome sequencing.

28. The method according to any one of claims 1 to 10 and 18 to 25, wherein the amount of DNA or RNA is measured by quantitative PCR, real-time PCR, digital PCR or sequencing.

29. The method according to claim 28, wherein the sequencing includes next-generation whole-genome sequencing.

30. The method according to any one of claims 1 to 10 and 18 to 25, wherein the amount of RNA or cell-free DNA is measured by using a microarray.

31. The method according to any one of claims 1 to 10 and 18 to 25, wherein the amount of donor-derived RNA or cell-free DNA is determined by using ratio-based and / or machine learning-artificial intelligence comparisons at a single or multiple time points.

32. The method according to any one of claims 1 to 10 and 18 to 25, wherein the amount of RNA or cell-free DNA is measured by using molecular barcodes and microscopy imaging.

33. The method according to any one of claims 1 to 10 and 18 to 32, wherein the target loci contain single nucleotide polymorphisms (SNPs).

34. The method according to any one of claims 1 to 10 and 18 to 33, wherein the cut-off threshold is the estimated percentage of donor-derived cell-free DNA or RNA in the total cell-free DNA or RNA or a function thereof.

35. The method according to any one of claims 1 to 10 and 18 to 34, wherein the cut-off threshold is proportional to the absolute concentration of donor-derived cell-free DNA or RNA.

36. The method according to any one of claims 1 to 10 and 18 to 34, wherein step (b) comprises 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 - 2000 pairs of forward and reverse PCR primers, for amplifying at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, at least 100 target loci, 2 - 10, 200 - 100, 50 - 500 or 50 - 2000 target loci.

37. The method according to any one of claims 1 to 10 and 18 to 34, wherein step (b) comprises using at least 100, at least 500, at least 1000, at least 2000, 10 - 1000, 100 - 10000, 50 - 50000, 500 - 20000 pairs of forward and reverse PCR primers, for multiplex amplifying at least 100, at least 500, at least 1000, at least 2000 target loci, 10 - 1000, 100 - 10000, 50 - 50000 or 500 - 20000 target loci.

38. The method according to any one of claims 6 to 10 and 22 to 37, wherein the RNA is cell-free RNA.

39. The method according to claim 37, wherein the cell-free RNA is derived from exosomes or microvesicles.

40. The method according to any one of claims 6 to 10 and 22 to 39, wherein the RNA is small messenger RNA (mRNA).

41. The method according to any one of claims 6 to 10 and 22 to 39, wherein the RNA is small non-coding RNA (sncRNA).

42. The method according to claim 39, wherein the sncRNA comprises microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA) or miscellaneous RNA (miscRNA).

43. The method according to any one of claims 6 to 10 and 22 to 42, which further comprises using CRISPR-Cas to target and deplete contaminating or excessive nucleic acid species in the sample, thereby increasing the fraction of desired reads mapping to the target loci of interest per sample and the sample throughput per sequencing run.

44. The method according to claim 43, wherein the sample comprises whole blood or hemolyzed contaminated blood, serum or plasma sample, and wherein multiple guide RNAs are used to target multiple loci in the same reaction to deplete contaminating or excessive nucleic acids, thereby increasing the detection rate of the target loci.

45. The method according to claim 43, wherein the contaminating or excessive nucleic acid species comprise hemoglobin mRNA, tRNA and rRNA, as well as miR-451, miR-144 and miR-486.

46. The method according to any one of claims 43 to 45, further comprising depleting adapter dimers, primer dimers, and unwanted ligation products from the amplified nucleic acid composition comprising the target locus, thereby increasing the fraction of desired reads mapping to the target locus of interest per sample and the sample throughput per sequencing run.

47. The method according to any one of claims 43 to 46, wherein a CRISPR-Cas system comprising Cas9 or Cas12 is utilized to remove nucleic acid species after reverse transcription of RNA and before multiplex amplification.

48. The method according to any one of claims 43 to 47, wherein a CRISPR-Cas system comprising Cas9 or Cas12 is utilized to remove nucleic acid species after 1 - 10 cycles of multiplex amplification of the complementary DNA.

49. The method according to any one of claims 43 to 48, wherein the contaminating or excessive nucleic acid species is RNA, and wherein a CRISPR-Cas system comprising Cas13 is used to remove contaminating or excessive RNA species from the sample.

50. The method according to any one of the preceding claims, wherein the xenograft recipient is a human subject.

51. The method according to any one of the preceding claims, wherein the xenograft recipient has received one or more xenograft organs selected from: pancreas, kidney, liver, heart, lung, intestine, thymus, and uterus.

52. The method according to any one of the preceding claims, wherein the sample is obtained from the xenograft recipient less than 18 months after transplantation.

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

54. The method according to claim 53, 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 xenograft recipient and / or xenograft donor, sex of the xenograft recipient and / or allograft donor.

55. The method according to any one of the preceding claims, wherein the xenograft is from a pig, primate, baboon, cow, or dog.

56. The method according to any one of the preceding claims, wherein the xenograft is from a pig.

57. The method according to any one of claims 1 to 10 and 18 to 56, wherein the cell-free DNA or RNA is derived from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenograft recipient.

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