Methods for determining and monitoring xenograft rejection by measuring nucleic acids or proteins derived from the xenograft - Patent Application 20070122999

Abstract

The present invention provides a method for preparing and analyzing a biological sample from a xenotransplant recipient, which method includes extracting fragmented or intact cell-free DNA, RNA (such as mRNA or miRNA), or protein from the xenotransplant recipient sample, and measuring the amount of cell-free DNA, RNA (such as mRNA or miRNA), or protein derived from the xenotransplant, thereby enabling assessment of xenotransplant rejection. Detection of cell-free DNA or RNA can be performed by preparing a sequencing library and performing whole-genome sequencing.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 413,738, filed October 6, 2022, which is incorporated herein by reference in its entirety.

[0002] Rapid detection of graft damage and / or rejection remains a challenge for transplant recipients. Xenotransplantation, the transplantation of 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 bring this approach to the clinic, including the production of genetically modified donor animal strains with reduced immunogenicity and a lower risk of animal virus transmission to humans, and clinical testing of animal organs for transplantation into human patients. Rapid advances in this research and the severe shortage of human-derived organs for transplantation may make xenotransplantation a primary approach to address end-stage organ failure in the clinic in the near future.

[0003] After xenotransplantation, tests for determining and monitoring transplant injury and / or rejection are important aspects of post-transplant care and determining the need for individualized immunosuppressive therapy. Conventional biopsy-based tests are invasive, costly, and can lead to delayed diagnosis of transplant injury and / or rejection. Therefore, there is a need for a non-invasive test for xenograft injury and / or rejection that is more sensitive and specific than conventional biopsy-based tests to enable earlier diagnosis of transplant injury and / or rejection.

[0004] Therefore, improved methods are needed for early and accurate diagnosis, screening, testing, and monitoring of xenograft injury and / or rejection. The present disclosure addresses this need. Summary of the Invention

[0005] In one aspect, the disclosure relates to a method for preparing a composition of DNA derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting cell-free DNA from a 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 xenotransplant, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection or damage.

[0006] In one aspect, the disclosure relates to a method for preparing a composition of amplified DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting cell-free DNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA includes cell-free DNA from the donor of the xenotransplant and cell-free DNA from the recipient; (b) performing multiplex targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume to prepare a composition of amplified DNA and detect and quantify the amount of cell-free DNA from the animal donor, wherein the target loci include a set of animal target loci and a set of human target loci; and (c) determining whether the amount of cell-free DNA from the animal donor, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0007] In one aspect, the disclosure provides a method for preparing a composition of amplified DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, 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; and (b) performing targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume, thereby preparing the composition of amplified DNA. the target loci include both human and animal target loci; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount 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 the human reads and the animal reads are distinguished based on the insertion sequence; and (c) determining whether the proportion of cell-free DNA derived from the animal donor or a function thereof exceeds a cutoff threshold indicative of xenotransplant rejection.

[0008] In one aspect, the disclosure relates to a method for preparing a composition of amplified DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting cell-free DNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing the composition of amplified DNA by performing targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume, wherein the target loci include one or more target loci indicative of xenotransplant rejection; and (c) determining the abundance of the one or more target loci indicative of xenotransplant rejection and determining whether the abundance, or a function thereof, of the one or more target loci indicative of xenotransplant rejection exceeds a cutoff threshold for indicative of xenotransplant rejection.

[0009] In one aspect, the present disclosure relates to a method for preparing a composition of DNA derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for assessing xenotransplant rejection, wherein the composition of DNA comprises one or more target loci indicative of xenotransplant rejection, and the assessing step further comprises determining the amount of the one or more target loci indicative of xenotransplant rejection and determining whether the amount or a function thereof of the one or more target loci indicative of xenotransplant rejection exceeds a cutoff threshold for indicative of xenotransplant rejection, wherein xenotransplant rejection is assessed by a combination of (i) the amount or a function thereof of the one or more target loci indicative of xenotransplant rejection and (ii) the total amount of cell-free DNA from the animal donor or a percentage of cell-free DNA from the animal donor.

[0010] In one aspect, the disclosure herein relates to a method for preparing a complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting RNA from a 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; and (c) determining whether the total amount of donor-derived RNA from the xenotransplant, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0011] In one aspect, the disclosure herein relates to a method for preparing a composition of amplified complementary DNA (cDNA) from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting RNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA from the xenotransplant; (b) preparing a composition of amplified cDNA from the extracted RNA by performing multiplex targeted amplification of cDNA at 10 to 50,000 target loci in a single reaction volume, and detecting and quantifying the amount of animal donor-derived RNA, wherein the target loci comprise a set of animal target loci and a set of human target loci; and (c) determining whether the amount of donor-derived RNA target loci, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0012] In one aspect, the disclosure provides a method for preparing a composition of amplified complementary DNA (cDNA) from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, comprising: (a) extracting RNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; and (b) performing targeted amplification of cDNA at 10 to 50,000 target loci in a single reaction volume, thereby preparing a composition of amplified cDNA derived from the extracted RNA. wherein the target loci include both human loci and target loci; sequencing the amplified cDNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of both donor-derived RNA and xenotransplant recipient-derived RNA based on the sequencing reads, wherein the human loci and the animal loci are the same and the human reads and the animal reads are distinguished based on the insert sequence; and (c) determining whether the proportion of donor-derived RNA or a function thereof exceeds a cutoff threshold indicative of xenotransplant rejection.

[0013] In one aspect, the disclosure herein relates to a method for preparing a composition of amplified complementary DNA (cDNA) from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for assessing xenotransplant rejection, the method comprising: (a) extracting RNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises cell-free RNA derived from the donor and RNA derived from the recipient; (b) preparing a composition of amplified cDNA derived from the extracted RNA by performing targeted amplification of cDNA at 10 to 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 abundance of the one or more target loci indicative of xenotransplant rejection and determining whether the abundance of, or a function thereof, of the one or more target loci indicative of xenotransplant rejection exceeds a cutoff threshold for indicative of xenotransplant rejection.

[0014] In one aspect, the disclosure herein relates to a method for preparing a composition of complementary DNA (cDNA) amplified from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, useful for assessing xenograft rejection, wherein the composition comprises target loci indicative of xenograft rejection, and the assessing step further comprises determining the amount of 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 cutoff threshold indicative of xenograft rejection, wherein xenograft rejection is assessed 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 derived from the animal donor or a proportion of RNA derived from the animal donor.

[0015] In one aspect, the disclosure relates to a method for preparing a composition of proteins from a blood, plasma, serum, or urine sample of a xenotransplant recipient useful for determining xenotransplant rejection, the method comprising: (a) extracting proteins from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the proteins include donor-derived proteins and recipient-derived proteins from the xenotransplant; (b) detecting and quantifying the amount of donor-derived proteins; and (c) determining whether the amount of donor-derived proteins or a function thereof exceeds a cutoff threshold indicative of xenotransplant rejection.

[0016] In one aspect, the disclosure relates to a method for preparing a composition of proteins from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting proteins from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the proteins include proteins derived from the donor and proteins derived from the xenotransplant; (b) detecting and quantifying the amount of one or more target proteins, wherein the one or more target proteins include both proteins derived from the recipient and proteins derived from the donor, wherein the one or more target proteins are indicative of xenotransplant rejection; and (c) determining whether the amount of the one or more target proteins, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0017] In one aspect, the disclosure relates to a method of administering immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of a donor-derived protein described herein; and (b) titrating the dosage of immunosuppressive therapy according to or a function of the amount of donor-derived protein.

[0018] In one aspect, the present disclosure relates to a method of administering immunosuppressive therapy in a xenograft recipient, the method comprising: (a) measuring the amount of one or more target proteins described herein; and (b) titrating the dosage of the immunosuppressive therapy according to or a function of the amount of the one or more target proteins.

[0019] In some embodiments, the methods herein further comprise repeating steps (a)-(b) longitudinally on the same xenograft recipient and determining longitudinal changes in the amount of the donor-derived protein, the donor-derived target protein, or a function thereof, and longitudinal changes in the amount of the donor-derived protein, the target protein, or a function thereof.

[0020] In some embodiments, the methods of the invention further comprise titrating the dosage of immunosuppressive therapy in response to longitudinal changes in the donor-derived protein, donor-derived target protein, or function thereof.

[0021] In some embodiments, the protein is derived from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of the xenotransplant recipient, comprising extracting the protein from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of the xenotransplant recipient.

[0022] In one aspect, the disclosure relates to a method of administering 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; (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 dosage of immunosuppressive therapy according to, or a function of, the amount of cell-free DNA and the amount of donor-derived cell-free DNA.

[0023] In some embodiments, the amount of donor-derived cell-free DNA is measured by extracting cell-free DNA from a blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification of the extracted DNA at 200 to 50,000 target loci in a single reaction volume; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads.

[0024] In some embodiments, the methods herein further include repeating steps (a)-(b) longitudinally 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.

[0025] In some embodiments, the methods herein further comprise titrating the dosage of 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.

[0026] In one aspect, the disclosure relates to a method of administering 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 the blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dosage of immunosuppressive therapy according to, or a function of, the amount of cell-free DNA and the amount of donor-derived RNA.

[0027] In some embodiments, the amount of donor-derived RNA is measured by extracting RNA from a blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted RNA includes donor-derived RNA and recipient-derived RNA; preparing a composition of amplified complementary DNA (cDNA) derived from the extracted RNA by performing multiplexed targeted amplification of cDNA at 200 to 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.

[0028] In some embodiments, the methods herein further include repeating steps (a)-(b) longitudinally on the same transplant recipient and determining longitudinal changes in the amount of RNA or a function thereof and longitudinal changes in the amount of donor-derived RNA or a function thereof.

[0029] In some embodiments, the methods herein further comprise titrating the dosage of immunosuppressive therapy according to 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.

[0030] In some embodiments, an internal control is added to the sample.

[0031] In some embodiments, the sequencing comprises shotgun whole genome sequencing.

[0032] In some embodiments, the amount of DNA or RNA is measured by quantitative PCR, real-time PCR, digital PCR, or sequencing.

[0033] In some embodiments, the sequencing comprises next-generation whole genome sequencing.

[0034] In some embodiments, the amount of RNA or cell-free DNA is measured by using a microarray.

[0035] In some embodiments, the amount of donor-derived RNA or cell-free DNA is determined by using ratiometric and / or machine learning artificial intelligence comparisons at single or multiple time points.

[0036] In some embodiments, the amount of RNA or cell-free DNA is measured by using molecular barcodes and microscopic imaging (such as NanoString NCOUNTER®).

[0037] In some embodiments, the target locus comprises a single nucleotide polymorphism (SNP).

[0038] In some embodiments, the cutoff threshold is an estimated ratio of donor-derived cell-free DNA or RNA to total cell-free DNA or RNA, or a function thereof.

[0039] In some embodiments, the cutoff threshold is proportional to the absolute value of the cell-free DNA or RNA concentration from the donor.

[0040] In some embodiments, step (b) comprises amplifying at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100 target loci from 2 to 10, 200 to 100, 50 to 500, or 50 to 2000 target loci using at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100 target RNA molecules from 2 to 10, 200 to 100, 50 to 500, or 50 to 2000 pairs of forward and reverse PCR primers.

[0041] Step (b) comprises multiplex amplification of at least 100, at least 500, at least 1000, or at least 2000 target loci from 10-1000, 100-10000, 50-50000, or 500-20000 target loci using at least 100, at least 500, at least 1000, or at least 2000 from 10-1000, 100-10000, 50-50000, or 500-20000 pairs of forward and reverse PCR primers.

[0042] In some embodiments, the RNA is cell-free RNA.

[0043] In some embodiments, the cell-free RNA is derived from exosomes or microvesicles.

[0044] In some embodiments, the RNA is a small messenger RNA (mRNA).

[0045] In some embodiments, the RNA is a small non-coding RNA (sncRNA).

[0046] In some embodiments, the sncRNA comprises a microRNA (miRNA), a piwi-interacting RNA (piRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), or other RNA (miscRNA).

[0047] In some embodiments, the methods herein further comprise utilizing CRISPR-Cas to target and deplete contaminating or excess nucleic acid species in a sample, thereby increasing the proportion of desired reads that map to target loci of interest per sample and sample throughput per sequencing run.

[0048] In some embodiments, the sample comprises whole blood or hemolyzed contaminated blood, serum, or plasma samples, and multiple guide RNAs are used to target multiple loci in the same reaction to deplete contaminating or excess nucleic acids, thereby increasing the detection rate of the target loci.

[0049] In some embodiments, the contaminating or excess nucleic acid species include hemoglobin mRNA, tRNA, rRNA, miR-451, miR-144, and miR-486.

[0050] In some embodiments, the methods herein further comprise depleting adapter dimers, primer dimers, and unwanted ligation products from a composition of amplified nucleic acid comprising a target locus, thereby increasing the proportion of desired reads that map to target loci of interest per sample and sample throughput per sequencing run.

[0051] In some embodiments, Cas9 / Cas12a is used to remove nucleic acid species after reverse transcription of RNA and before multiplex amplification.

[0052] In some embodiments, Cas9 / Cas12a is used to remove nucleic acid species after 1 to 10 cycles of multiplex amplification of complementary DNA.

[0053] In some embodiments, the contaminating or excess nucleic acid species is RNA, and Cas13 is used to remove the contaminating or excess RNA species from the sample.

[0054] In some embodiments, the xenograft recipient is a human subject.

[0055] In some embodiments, the xenograft recipient receives one or more xenograft organs selected from pancreas, kidney, liver, heart, lung, intestine, thymus, and uterus.

[0056] In some embodiments, the sample is obtained from the xenograft recipient within 18 months of transplantation.

[0057] In some embodiments, the risk of rejection of a xenotransplant recipient is determined using logistic regression, random forest, or decision tree machine learning analysis.

[0058] In some embodiments, the logistic regression, random forest, or decision tree machine learning analysis further incorporates one or more parameters selected from time since transplant, age of the xenograft recipient and / or xenograft donor, and gender of the xenograft recipient and / or xenograft donor.

[0059] In some embodiments, the xenograft is derived from a pig, a primate, a baboon, a cow, or a dog.

[0060] In some embodiments, the cell-free DNA or RNA is derived from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenotransplant recipient. [Brief explanation of the drawings]

[0061] [Figure 1] A workflow for testing a method to detect cell-free DNA from animal donors in human plasma samples by using a whole genome sequencing approach. [Figure 2] 1 is a graph showing the detection of cell-free DNA from animal donors in human plasma samples. The graph shows a linear relationship between the known amount of cell-free DNA from spiked animals and the amount measured using whole genome sequencing, thereby demonstrating proof of concept for a method of measuring cell-free DNA from animal donors in human plasma samples using whole genome sequencing. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present disclosure relates to methods for determining and monitoring xenograft rejection in human recipients based on the use of whole genome sequencing of cell-free DNA or RNA from blood, plasma, serum, or urine samples from the xenograft recipient. Alternatively, the present disclosure relates to methods for determining and monitoring xenograft rejection in human recipients based on measuring proteins derived from blood, plasma, serum, or urine samples from the xenograft recipient. In some embodiments, the cellular DNA, RNA, or proteins are isolated from extracellular vesicles (EVs) isolated from blood, plasma, serum, or urine samples from the xenograft recipient. Examples presented herein demonstrate that the presently disclosed methods can be used to detect animal-derived nucleic acids in human plasma samples.

[0063] A method for determining and monitoring xenograft rejection based on the measurement of cell-free DNA. In one aspect, the present disclosure relates to a method for preparing a composition of DNA derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for assessing 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 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 xenotransplant, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection or damage. In some embodiments, no amplification or pre-amplification is performed on the extracted cell-free DNA prior to sequencing. In some embodiments, preparing the sequencing library comprises adding adapters to the extracted cell-free DNA, for example, by ligation. In some embodiments, adding adapters to the extracted cell-free DNA comprises end repair, adding adenosines to the cell-free DNA fragments, followed by cohesive end ligation to the cell-free DNA fragments. In some embodiments, the cell-free DNA fragments are repaired and the generated blunt ends are filled in. In some embodiments, adding adapters to the extracted cell-free DNA comprises blunt end ligation of adapters to the cell-free DNA fragments.

[0064] In one aspect, the disclosure relates to a method for preparing a composition of amplified DNA derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting cell-free DNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA comprises cell-free DNA derived from the donor and cell-free DNA derived from the recipient of the xenotransplant; (b) preparing the composition of amplified DNA by performing multiplex targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume to detect and quantify the amount of cell-free DNA derived from the animal donor, wherein the target loci comprise a set of animal target loci and a set of human target loci; and (c) determining whether the amount of cell-free DNA derived from the animal donor, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0065] In one aspect, the disclosure provides a method for preparing a composition of amplified DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, 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; and (b) performing targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume, thereby preparing the composition of amplified DNA. the target loci include both human and animal target loci, sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of both donor-derived cell-free DNA and xenotransplant recipient-derived cell-free DNA based on the sequencing reads, wherein the human and animal loci are the same and the human and animal reads are distinguished based on an insertion sequence; and (c) determining whether the proportion of animal donor-derived cell-free DNA or a function thereof exceeds a cutoff threshold indicative of xenotransplant rejection. As used herein, "insertion sequence" refers to any sequence that differs in a host human target locus compared to the same animal target locus.

[0066] In one aspect, the disclosure relates to a method for preparing a composition of amplified DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting cell-free DNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted cell-free DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; (b) preparing the composition of amplified DNA by performing targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume, wherein the target loci include one or more target loci indicative of xenotransplant rejection; and (c) determining the abundance of the one or more target loci indicative of xenotransplant rejection and determining whether the abundance, or a function thereof, of the one or more target loci indicative of xenotransplant rejection exceeds a cutoff threshold for indicative of xenotransplant rejection.

[0067] In one aspect, the present disclosure relates to a method for preparing a composition of DNA derived from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for assessing xenotransplant rejection, wherein the composition of DNA comprises one or more target loci indicative of xenotransplant rejection, and the assessing step further comprises determining the amount of the one or more target loci indicative of xenotransplant rejection and determining whether the amount or a function thereof of the one or more target loci indicative of xenotransplant rejection exceeds a cutoff threshold for indicative of xenotransplant rejection, wherein xenotransplant rejection is assessed by a combination of (i) the amount or a function thereof of the one or more target loci indicative of xenotransplant rejection and (ii) the total amount of cell-free DNA from the animal donor or a percentage of cell-free DNA from the animal donor.

[0068] 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.

[0069] A method for determining and monitoring xenograft rejection based on RNA measurements. In one aspect, the present disclosure relates to a method for preparing a complementary DNA (cDNA) composition from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, the method being useful for assessing xenotransplant rejection, the method comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA includes donor-derived RNA and recipient-derived RNA; (b) preparing a cDNA sequencing library from the extracted RNA, 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; and (c) determining whether the total amount of donor-derived RNA from the xenotransplant, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection. In some embodiments, no amplification or pre-amplification is performed on the extracted RNA prior to sequencing. In some embodiments, preparing the sequencing library comprises adding adapters to the cDNA, for example, by ligation. In some embodiments, the cDNA fragments are repaired and the generated blunt ends are filled in. In some embodiments, adaptors are added to the cDNA fragments by blunt-end ligation. In some embodiments, adaptors are added to the cDNA by sticky-end ligation to generate a cDNA sequencing library.

[0070] In one aspect, the disclosure herein relates to a method for preparing a composition of amplified complementary DNA (cDNA) from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting RNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA from the xenotransplant; (b) preparing a composition of amplified cDNA from the extracted RNA by performing multiplex targeted amplification of cDNA at 10 to 50,000 target loci in a single reaction volume to detect and quantify the amount of animal donor-derived RNA, wherein the target loci comprise a set of animal target loci and a set of human target loci; and (c) determining whether the amount of donor-derived RNA target loci, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0071] In one aspect, the present disclosure provides a method for preparing a composition of amplified complementary DNA (cDNA) from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, comprising: (a) extracting RNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived RNA and recipient-derived RNA; and (b) performing targeted amplification of cDNA at 10 to 50,000 target loci in a single reaction volume, thereby obtaining a composition of amplified cDNA derived from the extracted RNA. preparing a product, wherein the target loci include both human loci and target loci; sequencing the amplified cDNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of both donor-derived RNA and xenotransplant recipient-derived RNA based on the sequencing reads, wherein the human loci and the animal loci are the same and the human reads and the animal reads are distinguished based on the insert sequence; and (c) determining whether the proportion of donor-derived RNA or a function thereof exceeds a cutoff threshold indicative of xenotransplant rejection.

[0072] In one aspect, the disclosure herein relates to a method for preparing a composition of amplified complementary DNA (cDNA) from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for assessing xenotransplant rejection, the method comprising: (a) extracting RNA from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises cell-free RNA derived from the donor and RNA derived from the recipient; (b) preparing a composition of amplified cDNA derived from the extracted RNA by performing targeted amplification of cDNA at 10 to 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 abundance of the one or more target loci indicative of xenotransplant rejection and determining whether the abundance of, or a function thereof, of the one or more target loci indicative of xenotransplant rejection exceeds a cutoff threshold for indicative of xenotransplant rejection.

[0073] In one aspect, the disclosure herein relates to a method for preparing a composition of amplified complementary DNA (cDNA) extracted from RNA extracted from a blood, plasma, serum, or urine sample of a xenograft recipient, useful for assessing xenograft rejection, wherein the composition comprises target loci indicative of xenograft rejection, and the assessing step further comprises determining the amount of one or more target loci indicative of xenograft rejection and determining whether the amount or function thereof of the one or more target loci indicative of xenograft rejection exceeds a cutoff threshold indicative of xenograft rejection, wherein xenograft rejection is assessed by a combination of the amount or function thereof of the one or more target loci indicative of xenograft rejection and the total amount of RNA derived from the animal donor or a proportion of RNA derived from the animal donor.

[0074] In some embodiments, the RNA is derived from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenotransplant recipient.

[0075] Methods for determining and monitoring xenograft rejection based on protein measurements. In one aspect, the disclosure relates to a method for preparing a composition of proteins from a blood, plasma, serum, or urine sample of a xenotransplant recipient useful for determining xenotransplant rejection, the method comprising: (a) extracting proteins from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the proteins include donor-derived proteins and recipient-derived proteins from the xenotransplant; (b) detecting and quantifying the amount of donor-derived proteins; and (c) determining whether the amount of donor-derived proteins or a function thereof exceeds a cutoff threshold indicative of xenotransplant rejection.

[0076] In one aspect, the disclosure relates to a method for preparing a composition of proteins from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting proteins from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the proteins include proteins derived from the donor and proteins derived from the xenotransplant; (b) detecting and quantifying the amount of one or more target proteins, wherein the one or more target proteins include both proteins derived from the recipient and proteins derived from the donor, wherein the one or more target proteins are indicative of xenotransplant rejection; and (c) determining whether the amount of the one or more target proteins, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0077] In one aspect, the present disclosure relates to a method for preparing a composition of proteins from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting proteins from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the proteins include donor-derived proteins and recipient-derived proteins from the xenotransplant; (b) detecting and quantifying the amount of donor-derived proteins; and (c) determining whether the amount of donor-derived proteins or a function thereof exceeds a cutoff threshold indicative of xenotransplant rejection.

[0078] In one aspect, the disclosure relates to a method for preparing a composition of proteins from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, the method comprising: (a) extracting proteins from extracellular vesicles (EVs) isolated from a blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the proteins include donor-derived proteins and recipient-derived proteins from the xenotransplant; (b) detecting and quantifying the amount of one or more target proteins, wherein the one or more target proteins include both recipient-derived proteins and donor-derived proteins, wherein the one or more target proteins are indicative of xenotransplant rejection; and (c) determining whether the amount of the one or more target proteins, or a function thereof, exceeds a cutoff threshold indicative of xenotransplant rejection.

[0079] In one aspect, the disclosure relates to a method of administering immunosuppressive therapy in a xenotransplant recipient, the method comprising: (a) measuring the amount of donor-derived protein; and (b) titrating the dosage of immunosuppressive therapy according to the amount of donor-derived protein or a function thereof.

[0080] In one aspect, the disclosure relates to a method of administering immunosuppressive therapy in a xenograft recipient, the method comprising: (a) measuring the amount of one or more target proteins; and (b) titrating the dosage of the immunosuppressive therapy according to or a function of the amount of the one or more target proteins.

[0081] In some embodiments, the methods herein further comprise repeating steps (a)-(b) longitudinally on the same xenograft recipient and determining longitudinal changes in the amount of the donor-derived protein, the donor-derived target protein, or a function thereof, and longitudinal changes in the amount of the donor-derived protein, the target protein, or a function thereof.

[0082] In some embodiments, the methods herein further comprise titrating the dosage of immunosuppressive therapy in response to longitudinal changes in the donor-derived protein, donor-derived target protein, or their functions.

[0083] Methods for measuring the amount of protein 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 protein of the present invention is, for example, an antibody capable of binding to the protein, preferably an antibody bearing a detectable label. The antibody can be polyclonal, or preferably monoclonal. An intact antibody or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeling" is intended to encompass both direct labeling of an antibody by binding a detectable substance to the antibody, and indirect labeling of an antibody by reactivity with another directly labeled reagent.

[0084] Various formats can be used to determine whether a sample contains a protein that binds to a specific antibody. Examples of such formats include, for example, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, Western blot analysis, and ELISAs. Numerous formats of antibody arrays have been proposed and described using antibodies. Such arrays typically contain different antibodies with specificity for different proteins intended to be detected. For example, typically, at least 100 different antibodies are used to detect 100 different protein targets, with each antibody specific for one target. In some embodiments, the amount of protein is measured using a mass spectrometry-based approach. In a related aspect, the present invention provides an array comprising a support(s) bearing multiple ligands that specifically bind to multiple proteins. The multiple proteins include at least two, three, four, or five proteins determined to be indicative of transplant rejection. In some embodiments, the number of proteins in the multiple proteins is less than 1000 or less than 100, and more than 100 or more than 10, respectively. In some embodiments, the multiple ligands are bound to a planar support or beads. In some embodiments, the ligands are different antibodies, and the different antibodies bind to different proteins in the multiple proteins.

[0085] In some embodiments, the target protein is encoded by an RNA target disclosed elsewhere herein.

[0086] Samples containing nucleic acids and methods for obtaining samples and extracting nucleic acids - Patents.com 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 derived from a pig, a primate, a baboon, a cow, or a dog.

[0087] In some embodiments, the xenotransplant recipient receives multiple transplanted organs selected from pancreas, kidney, liver, lung, heart, intestine, thymus, or uterus. In some embodiments, one or more transplanted organs are from the same transplant donor. In some embodiments, one or more transplanted organs are from more than one different transplant donor. In some embodiments, the transplant recipient receives multiple organs simultaneously.

[0088] 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.

[0089] In some embodiments, the sample is obtained from a xenograft recipient less than 18 months post-transplant, less than 17 months post-transplant, less than 16 months post-transplant, less than 15 months post-transplant, less than 14 months post-transplant, less than 13 months post-transplant, or less than 12 months post-transplant, hi some embodiments, the sample is obtained from a transplant recipient between 0-2 months post-transplant, 2-4 months post-transplant, 4-6 months post-transplant, 6-9 months post-transplant, 9-12 months post-transplant, or 12-18 months post-transplant.

[0090] 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 and determining longitudinal changes 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.

[0091] 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 multiple simultaneous organ transplants.

[0092] The sample may be a bodily fluid sample, tissue, organ, or individual cells. 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 may be extracellular vesicles derived from a bodily fluid sample, such as blood, plasma, serum, CSF, or urine.

[0093] Nucleic acids and methods for extracting or concentrating nucleic acids The methods disclosed herein include extracting nucleic acids from a sample derived from a subject. The nucleic acids can be cell-free DNA, intracellular DNA, DNA extracted from exosomes, cell-free RNA, intracellular 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, cellular, 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 other RNA (miscRNA). In some embodiments, the cell-free sncRNA is derived from exosomes or microvesicles.

[0094] In some embodiments, nucleic acids are extracted by size exclusion. In some embodiments, cell-free DNA or RNA is isolated from cellular DNA or RNA based on size. In some embodiments, nucleic acids are isolated by using affinity chromatography.

[0095] In some embodiments, nucleic acids are preferentially enriched. Nucleic acids may be preferentially enriched by using preferential enrichment at loci or target sites. Such preferential enrichment refers to any method whereby the proportion of nucleic acid molecules corresponding to loci in a nucleic acid mixture after enrichment is higher than the proportion of nucleic acid molecules corresponding to loci in the nucleic acid mixture before enrichment. The method may include selective amplification of nucleic acid molecules corresponding to loci. The method may include removing nucleic acid molecules that do not correspond to loci. The method may include a combination of methods. Enrichment is defined as the proportion of nucleic acid molecules corresponding to loci or targets in the mixture after enrichment divided by the proportion of nucleic acid molecules corresponding to loci or targets in the mixture before enrichment. Preferential enrichment may be performed at multiple loci. In some embodiments of the present disclosure, the enrichment is greater than 20. In some embodiments of the present disclosure, the enrichment is greater than 200. In some embodiments of the present disclosure, the enrichment is greater than 2,000. When preferential enrichment is performed at multiple loci, the enrichment may refer to the average enrichment of all loci in the set of loci.

[0096] Amplification refers to techniques that increase the copy number of a nucleic acid molecule. Selective amplification can refer to techniques that increase the copy number of a specific nucleic acid molecule or a nucleic acid molecule corresponding to a specific region of a nucleic acid molecule. It can also refer to methods that increase the copy number of a specific target molecule or target region of a nucleic acid molecule over the copy number of non-target molecules or regions of a nucleic acid molecule.

[0097] Selective amplification can be a preferential enrichment method. A universal priming sequence refers to a DNA sequence that can be added to a population of target DNA molecules, for example, by ligation, PCR, or ligation-mediated PCR. Once added to a population of target molecules, primers specific to the universal priming sequence can be used to amplify the target population using a single amplification primer pair. The universal priming sequence is typically not related to the target sequence. A universal adapter, or "ligation adapter" or "library tag," is a DNA molecule containing a universal priming sequence that can be covalently attached to the 5' and 3' ends of a population of target double-stranded DNA molecules. Addition of the adapter provides universal priming sequences at the 5' and 3' ends of the target population, where PCR amplification can be performed, amplifying all molecules from the target population using a single amplification primer pair. Targeting refers to a method used to selectively amplify or otherwise preferentially enrich molecules of DNA corresponding to a set of loci in a mixture of DNA.

[0098] Specific nucleic acids may also be enriched using hybrid capture. In some embodiments, preferentially enriching RNA with a plurality of biomarkers comprises obtaining a set of hybrid capture probes, hybridizing the hybrid capture probes to RNA in the sample, and physically separating the hybridized RNA from unhybridized RNA from the sample of RNA. In some embodiments, preferentially enriching sncRNAs, such as miRNAs, with a plurality of biomarkers comprises obtaining a set of hybrid capture probes, hybridizing the hybrid capture probes to miRNAs in the sample, and physically separating the hybridized miRNAs from the sample of RNA from unhybridized RNA. In some embodiments, preferentially enriching preselected mRNAs comprises obtaining a set of hybrid capture probes, hybridizing the hybrid capture probes to mRNAs in the sample, and physically separating the hybridized mRNAs from the sample of RNA from unhybridized RNA.

[0099] In some embodiments, in the methods disclosed herein, DNA is preferentially enriched at target loci or biomarkers.

[0100] The term "biomarker" refers to a molecule that is an indicator of an abnormal biological state (e.g., a disease or disorder, or transplant rejection). For example, a biomarker can be (a) expressed at a higher or lower level, (b) have an altered ratio compared to another biomarker, (c) present at a higher or lower level, (d) a variant or mutant of a gene product, or (e) simply a gene product (i.e., RNA or protein) that is present or absent in a cell or tissue sample from a subject with or suspected of having a disease, compared to a non-diseased tissue or cell sample from a subject with or suspected of having a disease, or compared to a cell or tissue sample from a subject or pool of subjects without or not suspected of having a disease. In the context of transplantation, a biomarker can be indicative of poor donor organ health or transplant rejection. That is, one or more gene products are sufficiently specific to the test sample that one or more can be used to identify, predict, or detect the presence of transplant rejection, disease, disease risk, risk of a predetermined event, or change in disease state, or to inform appropriate or improved treatment regimens.

[0101] In some embodiments, one or more biomarkers are a genetic abnormality or a set of genetic abnormalities, which are used herein to refer to the amount and nucleic acid variants of nucleic acid-containing particles. Specifically, genetic abnormalities include, but are not limited to, overexpression of a gene (e.g., an oncogene) or a panel of genes, underexpression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or a panel of genes, alternative splice variants of a gene or a panel of genes, gene copy number variations (CNVs) (e.g., DNA double minute chromosomes), nucleic acid modifications (e.g., methylation, acetylation, and phosphorylation), single nucleotide polymorphisms (SNPs), chromosomal rearrangements (e.g., inversions, deletions, and duplications), and gene or gene panel mutations (insertion, deletion, duplication, missense, nonsense, synonymous, or any other nucleotide change) (which often ultimately affect the activity and function of the gene product, leading to alternative transcriptional splice variants and / or altered gene expression levels), or any combination thereof.

[0102] In some embodiments, preferentially enriching DNA in a sample at a plurality of polymorphic loci includes obtaining a plurality of pre-circularized probes, each probe targeting one of the polymorphic loci and the 3' and 5' ends of the probes designed to hybridize to a region of DNA separated from the polymorphic site at the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the pre-circularized probes to DNA from the sample; using a DNA polymerase to fill in the gaps between the ends of the hybridized probes; circularizing the pre-circularized probes; and amplifying the circularized probes.

[0103] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci includes obtaining a plurality of ligation-mediated PCR probes, each PCR probe targeting one of the polymorphic loci and wherein the upstream and downstream PCR probes are designed to hybridize to a DNA region on one strand of the DNA separated from the polymorphic site of the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the ligation-mediated PCR probes to DNA from the first sample; using a DNA polymerase to fill gaps between the ends of the ligation-mediated PCR probes; ligating the ligation-mediated PCR probes; and amplifying the ligated ligation-mediated PCR probes.

[0104] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci includes obtaining a plurality of hybrid capture probes that target the polymorphic loci, hybridizing the hybrid capture probes to DNA in the sample, and physically removing some or all of the unhybridized DNA from the first DNA sample.

[0105] In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping. In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping, and the length of the capture probes on either side can be selected from the group consisting of less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases. In some embodiments, the hybrid capture probes are designed to hybridize to regions that overlap the polymorphic site, and the plurality of hybrid capture probes includes at least two hybrid capture probes for each polymorphic locus, and each hybrid capture probe is designed to be complementary to a different allele at the polymorphic locus.

[0106] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci includes obtaining a plurality of inner forward primers, each primer targeting one of the polymorphic loci, the 3' end of the inner forward primer designed to hybridize to a DNA region upstream of the polymorphic site and separated from the polymorphic site by a small number of bases, wherein the small number of bases is selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, or 31-60 base pairs; and optionally, each primer targeting a region of the polymorphic locus. The method includes obtaining a plurality of inner reverse primers that target one of the loci, the inner reverse primers being designed such that the 3' ends of the inner reverse primers hybridize to a DNA region upstream of the polymorphic site and are separated from the polymorphic site by a small number of bases, where the small number of bases is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, or 31 to 60 base pairs; hybridizing the inner primers to the DNA; and amplifying the DNA using polymerase chain reaction to form an amplicon.

[0107] In some embodiments, the method also includes obtaining a plurality of outer forward primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA upstream of the inner forward primer; optionally obtaining a plurality of outer reverse primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA immediately downstream of the inner reverse primer; hybridizing the first primer to the DNA; and amplifying the DNA using polymerase chain reaction.

[0108] In some embodiments, the method also includes obtaining a plurality of outer reverse primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA immediately downstream of the inner reverse primer; optionally obtaining a plurality of outer forward primers, each targeting one of the polymorphic loci and designed to hybridize to a region of DNA upstream of the inner forward primer; hybridizing the first primer to the DNA; and amplifying the DNA using polymerase chain reaction.

[0109] In some embodiments, preparing the first sample further comprises adding universal adapters to 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 are less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp, some of which are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%.

[0110] In some embodiments, amplifying the DNA is carried out in one or more separate reaction volumes, each separate reaction volume comprising 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.

[0111] In some embodiments, preparing the sample further comprises dividing the sample into multiple portions, with the DNA in each portion preferentially enriched for a subset of the multiple polymorphic loci. In some embodiments, the inner primers are selected by identifying primer pairs that are likely to form undesired primer duplexes and removing at least one of the identified primer pairs that are likely to form undesired primer duplexes from the multiple primers. In some embodiments, the inner primers include a region designed to hybridize either upstream or downstream of the targeted polymorphic locus, and optionally include a universal priming sequence designed to enable PCR amplification. In some embodiments, at least some of the primers further include a random region that varies for each individual primer molecule. In some embodiments, at least some of the primers further include a molecular barcode.

[0112] In some embodiments, the method includes (a) performing a multiplex polymerase chain reaction (PCR) on a nucleic acid sample containing target loci to simultaneously amplify at least 1,000 different target loci in a single reaction volume using (i) at least 1,000 different primer pairs, or (ii) at least 1,000 target-specific primers and either a universal primer or a tag-specific primer, to generate amplification products containing target amplicons, and (b) sequencing the amplified products. In some embodiments, the method does not include using a microarray.

[0113] In some embodiments, the method includes (a) performing a multiplex polymerase chain reaction (PCR) on a cell-free DNA sample containing target loci to simultaneously amplify at least 1,000 different target loci in a single reaction volume using (i) at least 1,000 different primer pairs, or (ii) at least 1,000 target-specific primers and either a universal primer or a tag-specific primer, to generate amplification products containing target amplicons, and b) sequencing the amplified products. In some embodiments, the method does not include using a microarray.

[0114] In some embodiments, mRNA is isolated using a probe that hybridizes to the polyA tail of the mRNA molecule.

[0115] After blood collection and before nucleic acid extraction, blood cells in the blood sample may burst and release long DNA fragments into the sample, increasing the total amount of cell-free DNA (cfDNA) and background noise, distorting the detected dd-cfDNA percentage. To reduce such background noise, two specific enrichment methods for dd-cfDNA have been considered, based on the observation that dd-cfDNA is usually shorter than DNA shredded from blood cells of transplant recipients. In one embodiment, size selection is applied to select shorter cfDNA. In another embodiment, a universal amplification step is applied to reduce noise (e.g., before applying multiplex PCR), based on the hypothesis that short dd-cfDNA (often in mononucleosomal form) is amplified more efficiently than long DNA from transplant recipients.

[0116] Target genes and loci and protein targets The nucleic acids may include biomarkers indicative of an immune response or various diseases or conditions described elsewhere herein. In some embodiments, the target loci include one or more distinct sets of target loci. In some embodiments, the target loci include a set of human target loci and a set of animal target loci, where 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 the one or more human target loci can be distinguished from the corresponding animal loci by the insertion sequence.

[0117] In some embodiments, the method includes extracting fragmented or intact mRNA from a sample of a xenotransplant recipient, wherein the extracted mRNA includes mRNA from the donor and / or recipient, and wherein the mRNA comprises a plurality of biomarkers indicative of an immune response or a disease or disorder. In some embodiments, the biomarkers are indicative of an increased immune response. In some embodiments, the biomarkers are indicative of a decreased immune response. In some particular embodiments, sncRNA (e.g., miRNA) biomarkers are indicative of an increased immune response or a decreased immune response.

[0118] In some embodiments, the presently disclosed methods include pre-selecting an RNA target molecule. In some embodiments, the RNA target molecule comprises an RNA species known to be associated with assessing organ health. In some embodiments, the present disclosure provides methods for identifying an RNA target molecule associated with assessing organ health.

[0119] In some embodiments, the methods disclosed herein further comprise preferentially enriching the RNA for a plurality of biomarkers indicative of xenograft rejection, hi some embodiments, the RNA biomarkers are indicative of an increased immune response or a decreased immune response.

[0120] In some embodiments, the biomarker comprises a single nucleotide polymorphism (SNP) locus.

[0121] Samples and methods for isolating nucleic acids from samples In some embodiments, the nucleic acid sample includes fragmented or digested nucleic acids. In some embodiments, the nucleic acid sample includes DNA, such as genomic DNA, cDNA, cell-free DNA (cfDNA), cell-free mitochondrial DNA (cf mDNA), cell-free DNA derived from nuclear DNA (cf nDNA), cellular DNA, or mitochondrial DNA.

[0122] 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, two cells, three cells, four cells, five cells, six cells, seven cells, eight cells, nine cells, ten cells, or more than ten cells. In some embodiments, the nucleic acid sample is a substantially cell-free blood or plasma sample. In some embodiments, the nucleic acid sample comprises or is derived from blood, plasma, saliva, semen, sperm, cell culture supernatant, mucus secretion, dental plaque, gastrointestinal tissue, stool, urine, hair, bone, body fluids, tears, tissue, skin, nail, germ cells, embryos, amniotic fluid, chorionic villus samples, bile, lymph, cervical mucus, or a forensic sample. In some embodiments, the target locus is a fragment of a human nucleic acid. In some embodiments, the target locus is a fragment of human nucleic acid found 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.

[0123] In some embodiments, the method includes isolating or purifying DNA and / or RNA. There are several standard procedures known in the art for achieving such ends. In some embodiments, the sample may be centrifuged to separate the various layers. In some embodiments, DNA or RNA may be isolated using filtration. In some embodiments, DNA or RNA preparation may involve amplification, separation, chromatographic purification, liquid separation, isolation, preferential enrichment, preferential amplification, target amplification, or any of several other techniques known in the art or described herein. In some embodiments for DNA isolation, RNase is used to degrade RNA. In some embodiments for RNA isolation, DNase (such as DNase I from Invitrogen, Carlsbad, CA, USA) is used to degrade DNA. In some embodiments, the RNEASY™ Mini Kit (Qiagen) is used to isolate RNA according to the manufacturer's protocol. In some embodiments, small RNAs are isolated using the MIRVANA™ PARIS kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol (Gu et al., J. Neurochem. 122:641-649, 2012, incorporated herein by reference in its entirety). RNA concentration and purity may optionally be determined using Nanovue (GE Healthcare, Piscataway, NJ, USA), and RNA integrity may optionally be measured using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (Gu et al., J. Neurochem. 122:641-649, 2012, incorporated herein by reference in its entirety). In some embodiments, TRIZOL or RNALATER™ (Ambion) are used to stabilize RNA during storage.

[0124] In some embodiments, adapters are added to generate a sequencing library. Prior to ligation, the sample DNA may be blunt-ended, and then a single adenosine base is added to the 3' end. In some embodiments, ligation of the adapter to the nucleic acid is cohesive end ligation. Prior to ligation, the DNA may be cleaved using a restriction enzyme or some other cleavage method. During ligation, the 3'-terminal adenosine of the sample fragment and the complementary 3'-terminal tyrosine overhang of the adapter can increase ligation efficiency. In some embodiments, adapter ligation is performed using a ligation kit such as found in the AGILENT SURESELECT™ kit.

[0125] In some embodiments, the library is amplified using universal primers. In one embodiment, the amplified library is fractionated by size separation or by using products such as AGENCOURT AMPURE™ beads or other similar methods. In some embodiments, PCR amplification is used to amplify the target loci. In some embodiments, the amplified DNA is sequenced (such as by sequencing using an ILLUMINA IIGAX™ or HiSeq sequencer). In some embodiments, the amplified DNA is sequenced from each end of the amplified DNA to reduce sequencing errors. If a sequence error exists at a particular base when sequencing from one end of the amplified DNA, there is less likely to be a sequence error in the complementary base when sequencing from the other end of the amplified DNA (compared to multiple sequencing from the same end of the amplified DNA).

[0126] In some embodiments, miRNA can be separated from RNA fragments caused by degradation because degraded RNA loses terminal phosphorylation groups. The miRNA retains terminal phosphorylation groups. Adapters can ligate to phosphorylated miRNA ends, but adapters do not ligate to unphosphorylated RNA species, such as degraded mRNA. Adapters can include sequences that allow primer binding to support reverse transcription, selectively generating complementary DNA (cDNA).

[0127] As non-limiting examples, the locus may be a single nucleotide polymorphism, an intron, or an exon. In some embodiments, the locus may include an insertion, deletion, or rearrangement. In some embodiments, the sample may include a blood, serum, or plasma sample. In some embodiments, the sample may include free-floating DNA (e.g., circulating cell-free tumor DNA or circulating cell-free fetal DNA) in a blood, serum, or plasma sample. In these embodiments, the sample is typically from an animal, such as a mammal or human, and is typically present in fragments of approximately 160 nucleotides in length. In some embodiments, free-floating DNA is isolated from blood using EDTA-2Na tubes after centrifugation to remove cellular debris and platelets. Plasma samples can be stored at -80°C until DNA is extracted, for example, using a QIAAMP™ DNA Mini Kit (Qiagen, Hilden, Germany) (e.g., Hamakawa et al., Br J Cancer. 2015, 112:352-356). However, samples 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 mixed populations, particularly in environmental and biodiversity sampling.

[0128] Many kits and methods for generating libraries of nucleic acid molecules for subsequent sequencing are known in the art. Kits specifically adapted for preparing libraries from small nucleic acid fragments, particularly circulating cell-free DNA, can be useful for performing the methods provided herein. For example, the NEXTFLEX™ Cell Free Kit (Bioo Scientific, Austin, Texas) or the Natera Library Prep Kit (Natera, San Carlos, California). Such kits are typically modified to include adapters customized for the amplification and sequencing steps of the methods provided herein. Adapter ligation can also be performed using commercially available kits, such as the ligation kit found in the Agilent SURESELECT™ Kit (Agilent, Santa Clara, California).

[0129] The sample nucleic acid molecule is composed of natural or unnatural ribonucleotides or deoxyribonucleotides linked via phosphodiester bonds. Furthermore, the sample nucleic acid molecule is composed of nucleic acid fragments targeted for sequencing. The sample nucleic acid molecule can be or contain nucleic acid fragments at least 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides in length. In any of the embodiments disclosed herein, the sample nucleic acid molecules or nucleic acid fragments may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the lower end of the range and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the upper end of the range. can be between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length. In some embodiments, the nucleic acid molecule can be a fragment of genomic DNA and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the lower end of the range and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the upper end of the range. For clarity, nucleic acids initially isolated from biological tissues, fluids, or cultured cells can be much longer than the sample nucleic acid molecules processed using the methods herein.As discussed herein, for example, such initially isolated nucleic acid molecules can be fragmented to generate nucleic acid fragments before use in the methods herein. In some embodiments, the nucleic acid molecule and the nucleic acid fragment can be identical. The sample nucleic acid molecule or sample nucleic acid fragment can include a target locus that contains a nucleotide, i.e., a nucleotide(s) being queried, particularly a single nucleotide polymorphism or single nucleotide mutation. In any of the disclosed embodiments, the target locus can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides in length and can comprise a portion or the entire sample nucleic acid molecule and / or sample nucleic acid fragment. In other embodiments, the target locus is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length at the lower end of the range and 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length. In some embodiments, the target loci of different sample nucleic acid molecules can be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical. In some embodiments, target loci of different sample nucleic acid molecules can share at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity.

[0130] In some embodiments, the entire sample nucleic acid molecule is a sample nucleic acid fragment. For example, in certain embodiments where an adapter is directly ligated to the end of the sample nucleic acid molecule, or to a nucleic acid(s) ligated to the end of the sample nucleic acid molecule, or is ligated as part of a primer that binds to a sequence at the end of the sample nucleic acid fragment, or an adapter such as a universal adapter added thereto, as further described herein, the entire nucleic acid molecule can be a sample nucleic acid fragment. In other embodiments, for example, in certain embodiments where an adapter is added to the sample nucleic acid molecule as part of a primer that targets an internal binding site at the end of the sample nucleic acid molecule, a portion of the sample nucleic acid molecule can be a sample nucleic acid fragment targeted for downstream sequencing. For example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sample nucleic acid molecule can be a nucleic acid fragment.

[0131] In some embodiments, the sample nucleic acid molecules are a mixture of nucleic acids isolated from natural sources, with some sample nucleic acid molecules having identical sequences, some sharing at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity, and some having less than 50%, 40%, 30%, 20%, 10%, or 5% sequence identity across a range of 20, 25, 50, 75, 100, 125, 150, 200, or 250 nucleotides at the lower end to a range of 50, 75, 100, 125, 150, 200, 250, 300, 400, or 500 nucleotides at the upper end. Such sample nucleic acid molecules may be nucleic acid samples isolated from tissues or bodily fluids of mammals, such as humans, without enriching for certain sequences over others. In other embodiments, target sequences, e.g., from genes of interest, can be enriched prior to performing the methods provided herein.

[0132] Removal of contamination Biological samples can contain large amounts of contaminating nucleic acids, which can make target nucleic acid detection difficult, reduce the quality of nucleic acid libraries, and / or decrease assay throughput. For example, blood cells in a blood sample may rupture and release nucleic acids into the sample, resulting in a sample containing large amounts of unrelated nucleic acids forming red blood cells. In one embodiment of the present disclosure, a 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 target sequence-specific manner. The components required for in vitro digestion include a Cas effector protein and a target-specific guide RNA (gRNA).

[0133] An effective CRISPR-Cas complex requires several features. First, the gRNA must have a spacer element that is complementary to the target nucleic acid. This complementary target nucleic acid sequence is called a protospacer. Additionally, a protospacer adjacent motif (PAM) must be present immediately downstream of the spacer (for Cas9, immediately upstream of the spacer for Cas12a). For Cas9, this motif is NGG (where N is any nucleotide), whereas for Cas12a, the PAM motif can be TTTV (where V is any nucleotide except T). Proper complex formation also requires a gRNA scaffold. However, because this is conserved across all gRNAs for the same Cas enzyme, IDT standardized this for Cas9 and created a separate "tracrRNA" that anneals to a shorter "crRNA" to form the complete gRNA.

[0134] Various 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 that can then be repaired by non-homologous end joining or homologous recombination with donor template DNA, creating site-specific edits. Type II-A Cas9 generally has high genome editing efficiency but can suffer from off-target cleavage at unintended genomic sites. Variants have been designed to overcome these limitations, and type II-C Cas9 tends to have naturally high fidelity. Cas9 uses a guide spacer that is 18–24 nucleotides (nt) in length. The total length of the Cas9 guide can be approximately 100 nt. The PAM sequence can be 3-NGG (SpCas9), 3-NNGRRT (SaCas9), or 3-NNNNGATT (NmCas9). Cas9 generates blunt-ended dsDNA breaks. Mutant Cas9 enzymes that generate single-stranded nicks in one strand of the target DNA, as well as non-cleaving mutants that bind only to the target, are also commercially available.

[0135] Cas12 belongs to the Type V CRISPR-Cas family, including subtypes VA and VB, also known as Cpf1 (type VA) or C2c1 (type VB), among other subtypes. Cas12 is a compact and efficient enzyme that makes alternating cuts in dsDNA, thereby creating 3- to 5-base overhangs. The Cas12 guide spacer is 18-25 nt long, with the entire guide length being 42-44 nt. The Cas12 PAM sequence may be 5-TTTN (FnCas12a). Cas12 processes its own guide RNA, resulting in increased multiplexing capacity. Cas12 has also been engineered as a platform for epigenome editing, and it has recently been discovered that Cas12a can indiscriminately cleave single-stranded DNA when activated by a target DNA molecule matching the spacer sequence.

[0136] Cas13 is a type VI CRISPR-Cas, including subtypes VI-A, VI-B, VI-C, and VI-D, also known as C2c2 (VI-A) or CasRx (type VI-D). Cas13 targets RNA, not DNA. The Cas13 guide spacer is 22–30 nt long, with the entire guide length ranging from 52–66 nt. Cas13 PAM sequences include 3-H (LshCas13a), 5-D, 3-NAN or NNA (BzCas13b), and none (RfCas13d). When Cas13 is activated by a ssRNA sequence complementary to the crRNA spacer, it exhibits nonspecific RNase activity, destroying all nearby RNAs regardless of sequence. This property has been exploited in vitro for precision diagnostics. These systems can also be used for efficient, multiplexable specific RNA knockdown or RNA sequence editing in mammalian cells.

[0137] The methods disclosed herein can include utilizing any CRISPR-Cas system to deplete contaminating or excess 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 improving the sensitivity of detecting target loci. In another embodiment, Cas enzymes that target 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.

[0138] A mixture of different gRNAs can be used with the Cas enzyme to target many different loci in the same reaction. In some embodiments, one gRNA is used to target a locus. In some embodiments, one to five gRNAs are used to target one to five loci in the same reaction. In some embodiments, one to ten gRNAs are used to target one to ten loci in the same reaction. In some embodiments, one to one hundred gRNAs are used to target one to one hundred loci in the same reaction. In some embodiments, one to five,000 gRNAs are used to target one to five,000 loci in the same reaction. In some embodiments, ten to five,000 gRNAs are used to target one to five,000 loci in the same reaction. In some embodiments, one hundred to five,000 gRNAs are used to target one to five,000 loci in the same reaction. In some embodiments, 1,000 to 50,000 gRNAs are used to target 1,000 to 50,000 loci in the same reaction. In some embodiments, 10,000 to 5,000 gRNAs are used to target 10,000 to 50,000 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 1,000 gRNAs are used to target at least 1,000 loci in the same reaction. In some embodiments, at least 2,000 gRNAs are used to target at least 2,000 loci in the same reaction. In some embodiments, at least 3,000 gRNAs are used to target at least 3,000 loci in the same reaction. In some embodiments, at least 4,000 gRNAs are used to target at least 4,000 loci in the same reaction. In some embodiments, at least 5,000 gRNAs are used to target at least 5,000 loci in the same reaction. In some embodiments, at least 10,000 gRNAs are used to target at least 10,000 loci in the same reaction. In some embodiments, at least 20,000 gRNAs are used to target at least 20,000 loci in the same reaction.

[0139] Non-target miRNA analysis can be performed by ligating the miRNA to an adapter, as outlined above. The adapter may provide an NGG site adjacent to the miRNA to allow Cas9 cleavage. Once an unwanted sequence is ligated to the adapter, a Cas9-gRNA complex complementary to the unwanted sequence is introduced. This removes the adapter and prevents PCR amplification. This step is performed on double-stranded reverse-transcribed DNA.

[0140] In some embodiments, pilot sequencing runs can be used to learn which targets need to be removed from future sequencing runs. This allows assay developers to focus only on miRNAs of interest by designing gRNAs that target the most common uninteresting or contaminating small RNA fragments actually observed in the particular sample type used in the assay. This could include highly abundant miRNAs or small fragments of ribosomal or messenger RNA that may cause background noise.

[0141] In some embodiments, the Cas enzymes can be removed using heat-labile proteinase K (NEB P8111S), as Cas complexes are relatively long-lived and may interfere with downstream applications.

[0142] This approach may also be used for targeted miRNA applications, although it may be desirable to improve / remove bad primers prior to the assay rather than attempting to remove them after amplification using CRISPR-Cas.

[0143] In some embodiments, CRISPR-Cas-mediated removal of contaminating species can also be used in mRNA applications. In some embodiments, the CRISPR-Cas approach 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, approaches similar to those proposed for miRNAs can be used to design assays that specifically target the observed sequences. This is particularly useful, for example, when barcode primer dimers consume a large portion of the sequencing reads.

[0144] In some embodiments, a CRISPR-Cas approach is used to remove contaminating mRNA species greater than 20 nucleotides in length. In some embodiments, a CRISPR-Cas approach is used in combination with a "tag and capture" method to remove contaminating mRNA species greater than 20 nucleotides in length.

[0145] In some embodiments, CRISPR-Cas methods for removing contaminants are used on cDNA derived from miRNA or mRNA prior to amplification. In some embodiments, CRISPR-Cas methods for removing contaminants are used on cDNA derived from miRNA or mRNA, and the cDNA is amplified for 1 to 5 cycles, 1 to 10 cycles, or 1 to 15 cycles. In some embodiments, the cDNA is amplified for no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles prior to CRISPR-Cas-mediated removal of contaminants.

[0146] In some embodiments, the contaminating or excess species include hemoglobin mRNA, tRNA, rRNA, and miRNAs such as miR-451, miR-144, and miR-486, thereby increasing the proportion of desired reads that map to target loci of interest per sample and sample throughput per sequencing run.

[0147] In some embodiments, the method further comprises depleting adapter dimers, primer dimers, and unwanted ligation products from a composition of amplified nucleic acid comprising the target loci, thereby increasing the proportion of desired reads that map to target loci of interest per sample and sample throughput per sequencing run.

[0148] Methods for identifying biomarkers of interest for organ health transplantation Text mining databases identify known biomarkers of interest for transplant health. Biomarkers associated with transplant organ health are identified and common signatures are sought for a randomly selected set of biomarkers. Artificial intelligence may be used for text mining and prediction of known biomarkers of interest for transplant rejection and organ health.

[0149] Combining cell-free DNA and RNA measurements to assess and / or monitor transplant rejection In some embodiments, the methods herein further include (i) measuring the amount of donor-derived cell-free DNA in a sample obtained from the xenotransplant recipient and extracting cell-free DNA from the sample obtained from the xenotransplant recipient, wherein the extracted cell-free DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; (ii) performing targeted amplification of the extracted DNA at 50 to 50,000 target loci in a single reaction volume; (iii) sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, quantifying the amount of donor-derived cell-free DNA based on the sequencing reads, and determining xenotransplant rejection based on whether the amount of donor-derived cell-free DNA or a function thereof exceeds a cutoff threshold for cell-free DNA amount indicative of xenotransplant rejection, wherein xenotransplant rejection is determined based on whether both the amount of donor-derived RNA and the amount of donor-derived cell-free DNA, or a function thereof, exceed a cutoff threshold for xenotransplant rejection. The combination of the amount of RNA and cfDNA in a sample obtained from a xenotransplant recipient can serve as a biomarker for rejection and for the overall state of immunosuppression. In some embodiments, the combination of the amount of mRNA target selected from a group of preselected targets and the amount of cfDNA in the 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.

[0150] Determining the risk of rejection in transplant recipients In some embodiments, the risk of rejection of a xenotransplant recipient is determined using logistic regression, random forest, or decision tree machine learning analysis. In some embodiments, the machine learning analysis incorporates as a parameter the amount of donor-derived RNA in the transplant recipient sample, or a function thereof. In some embodiments, the machine learning analysis incorporates as a parameter the number of donor-derived RNA reads, or a function thereof. In some embodiments, the machine learning analysis incorporates as a parameter the estimated ratio of donor-derived RNA to total RNA. In some embodiments, the machine learning analysis incorporates as a parameter the amount of cell-free DNA, the number of cell-free DNA reads, or the estimated ratio of cell-free DNA to total cell-free DNA in the transplant recipient sample. In some embodiments, the machine learning analysis incorporates as a parameter the amount of a plurality of proteins derived from the xenotransplant. In some embodiments, the machine learning analysis further incorporates as a parameter the amount of total cell-free DNA in the transplant recipient sample, or a function thereof. In some embodiments, the machine learning analysis further incorporates as a parameter the number of total cell-free DNA reads, or a function thereof.

[0151] Machine learning may be used to determine whether a patient is rejected or not. Machine learning is disclosed in WO2020 / 018522, filed on July 16, 2019 as PCT / US2019 / 041981, entitled "Methods and Systems for Calling Ploidy States Using a Neural Network," the entire contents of which are incorporated herein by reference. In some embodiments, the cutoff threshold is scaled according to the amount of total cfDNA or RNA in the blood sample.

[0152] In some embodiments, the cutoff threshold is expressed as the percentage of dd-cfDNA in the blood sample (dd-cfDNA%). In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA per volume unit of blood sample. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of dd-cfDNA per volume unit of blood sample multiplied by the transplant recipient's body weight, BMI, or blood volume.

[0153] In some embodiments, the cutoff threshold takes into account the patient's weight, BMI, or blood volume. In some embodiments, the cutoff threshold takes into account one or more of donor genome copies per volume of plasma, cell-free DNA yield per volume of plasma, donor height, donor weight, donor age, donor sex, donor ethnicity, donor organ weight, donor organ, living vs. deceased donor, donor-recipient familial relationship (or lack thereof), recipient height, recipient weight, recipient age, recipient sex, recipient ethnicity, creatinine, eGFR (estimated glomerular filtration rate), cfDNA methylation, DSA (donor-specific antibodies), KDPI (Kidney Donor Profile Index), medications (immunosuppressants, steroids, anticoagulants, etc.), infections (BKV, EBV, CMV, UTI), recipient and / or donor HLA allele or epitope mismatch, Banff classification of renal allograft pathology, and cause-specific vs. surveillance or protocol biopsy.

[0154] In some embodiments, the method has a sensitivity of at least 50% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 60% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 70% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 80% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 85% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 90% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 95% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample.

[0155] In some embodiments, the method has a specificity of at least 50% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 60% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 70% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 75% and a confidence interval of 95% for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method is at least 80% specific and has a 95% confidence interval for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method is at least 85% specific and has a 95% confidence interval for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample. In some embodiments, the method is at least 90% specific and has a 95% confidence interval for identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample.In some embodiments, the method has a specificity of at least 95% with a 95% confidence interval in identifying AR versus non-acute rejection (AR) when the amount of dd-cfDNA is above a cutoff threshold scaled or adjusted according to the amount of total cfDNA in the blood sample.

[0156] 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 isolation of the cell-free DNA; b) performing target 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 obtained from the first tracer DNA composition.

[0157] Some embodiments use either a fixed threshold of donor DNA per plasma volume, or a non-fixed one, such as one adjusted or scaled as described herein. The method for determining this may be based on building an algorithm that maximizes performance using a training data set. Other data, such as patient weight, age, or other clinical factors, may also be considered.

[0158] In some embodiments, the method further includes using the amount of donor-derived cell-free DNA to determine the occurrence or likelihood of transplant rejection. In some embodiments, the amount of donor-derived cell-free DNA is compared to a cutoff threshold to determine the occurrence or likelihood of transplant rejection, the cutoff threshold being adjusted or scaled according to the amount of total cell-free DNA. In some embodiments, the cutoff threshold is a function of the number of donor-derived cell-free DNA reads.

[0159] In some embodiments, the method includes applying a scale or dynamic threshold metric that takes into account the amount of total cfDNA in the sample to more accurately assess transplant rejection. In some embodiments, the method further includes flagging the sample if the amount of total cell-free DNA is above a predetermined value. In some embodiments, the method further includes flagging the sample if the amount of total cell-free DNA is below a predetermined value.

[0160] RNA, DNA, or protein may be extracted from a sample from the transplant recipient, the sample including blood, plasma, serum, cerebrospinal fluid (CSF), or urine.

[0161] In some embodiments, the machine learning analysis further incorporates time since transplant as a parameter. In some embodiments, the machine learning analysis further incorporates age of the transplant recipient and / or transplant donor as a parameter. In some embodiments, the machine learning analysis further incorporates gender of the transplant recipient and / or transplant donor as a parameter.

[0162] In some embodiments, the risk of rejection of the transplant recipient is determined with a sensitivity of at least 0.81, or at least 0.82, or at least 0.83, or at least 0.84, or at least 0.85, or at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90. In some embodiments, the risk of rejection of the transplant recipient is determined with a specificity of at least 0.81, or at least 0.82, or at least 0.83, or at least 0.84, or at least 0.85, or at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90. In some embodiments, the risk of rejection for a transplant recipient is determined by an area under the curve (AUC) of at least 0.86, or at least 0.87, or at least 0.88, or at least 0.89, or at least 0.90, or at least 0.91, or at least 0.92, or at least 0.93, or at least 0.94, or at least 0.95.

[0163] Method for measuring the amount of nucleic acid 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.

[0164] In some embodiments, the amount of nucleic acid (e.g., RNA and / or DNA) from the donor is determined using a ratiometric and / or machine learning artificial intelligence comparison at single or multiple time points. In some embodiments, the amount of mRNA from the donor is determined using a ratiometric and / or machine learning artificial intelligence comparison at single or multiple time points. In some embodiments, the amount of miRNA from the donor is determined using a ratiometric and / or machine learning artificial intelligence comparison at single or multiple time points.

[0165] In some embodiments, the amount of RNA or cell-free DNA is measured by quantitative PCR. In some embodiments, the amount of mRNA is measured by quantitative PCR. In some embodiments, the amount of miRNA is measured by quantitative PCR. In some embodiments, the quantitative PCR includes real-time PCR or digital PCR.

[0166] In some embodiments, the amount of mRNA or cell-free DNA is measured by massively multiplexed PCR (mmPCR) to obtain amplicons containing the biomarkers and sequencing the amplicons.

[0167] In some embodiments, the amount of nucleic acid (eg, mRNA, miRNA, or cell-free DNA) is measured by using a microarray.

[0168] In some embodiments, the amount of nucleic acid (e.g., mRNA, miRNA, or cell-free DNA) is measured by using molecular barcodes and microscopic imaging (such as NanoString NCOUNTER®).

[0169] 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 the transplant recipient, wherein the extracted RNA includes donor-derived RNA and recipient-derived RNA; performing targeted amplification of the extracted RNA at 100 to 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, amplifying the RNA includes performing reverse transcriptase to obtain complementary DNA (cDNA).

[0170] 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 the transplant recipient, wherein the extracted cell-free DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification of the extracted DNA at 10 to 50,000 target loci in a single reaction volume; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads.

[0171] In some embodiments, the method is performed without prior knowledge of the donor genotype. In some embodiments, the method does not include determining the genotype of the transplant donor(s).

[0172] In some embodiments, the amount of nucleic acid is measured by target amplification. In some embodiments, the amount of a specific mRNA target is measured by target amplification. In some embodiments, the target amplification comprises PCR. In some embodiments, the primers for target amplification comprise 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 pairs of forward and reverse PCR primers. In some embodiments, target amplification is performed using 500 to 20,000, 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 primer pairs in a single reaction. This involves performing amplification at 100-20,000, 500-20,000, 1,000-10,000, 200-500, 500-1,000, 1,000-2,000, 2,000-5,000, 5,000-10,000, 10,000-20,000, and 20,000-50,000 target loci and obtaining amplification products.

[0173] In some embodiments, the target amplification comprises nested PCR. In some embodiments, the primers for target amplification comprise a first universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 targets. and a second universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 inner target-specific primers. In some embodiments, target amplification is performed using a first universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 target-specific primers. The method includes performing amplification at 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 target loci in a single reaction volume using a PCR product to obtain amplification products.In some embodiments, target amplification is performed using a second universal primer and 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 inner target-specific primers. Using primers, amplification is performed at 10 to 50,000, 100 to 50,000, 200 to 50,000, 500 to 20,000, or 1,000 to 10,000, 200 to 500, 500 to 1,000, 1,000 to 2,000, 2,000 to 5,000, 5,000 to 10,000, 10,000 to 20,000, or 20,000 to 50,000 target loci in a single reaction volume to obtain amplification products. In some embodiments, the methods disclosed herein comprise PCR amplification of at least 10, at least 100, at least 500, at least 1000, at least 2000 biomarkers from 10-1000, 100-10000, 200-50000, or 500-20000 RNA biomarkers using at least 10, at least 100, at least 500, at least 1000, at least 2000 from 10-1000, 100-10000, 200-50000, 500-20000 pairs of forward and reverse PCR primers. In some embodiments, step (b) comprises amplifying at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100 target RNA molecules from 2 to 10, 200 to 100, 50 to 500, or 50 to 2000 target RNA molecules using at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100 target RNA molecules from 2 to 10, 200 to 100, 50 to 500, or 50 to 2000 pairs of forward and reverse PCR primers.

[0174] In some embodiments, the method further comprises adding a tag to the amplification product prior to performing high-throughput sequencing, the tag comprising a sequencing-compatible adapter. In some embodiments, the method further comprises adding a tag to the extracted RNA prior to performing target amplification, the tag comprising an adapter for amplification. In some embodiments, the tag comprises a sample-specific barcode, and the method further comprises pooling the amplification products from multiple samples prior to high-throughput sequencing and sequencing the pool of amplification products together in a single run during high-throughput sequencing.

[0175] In some embodiments, the amount of nucleic acid is determined, for example, by using tracer nucleic acid or internal calibration nucleic acid. The terms "tracer nucleic acid" and "internal calibration nucleic acid" are used interchangeably and refer to the composition of nucleic acid, the length, sequence, nucleotide composition, amount, or biological origin of which are known in advance. The tracer can be added to a biological sample from a human subject to help estimate 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.

[0176] Cutoff threshold for determining transplant rejection In some embodiments, the cutoff threshold is an estimated percentage of donor-derived RNA relative to total RNA, or a function thereof. In some embodiments, the cutoff 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 (e.g., mRNA or miRNA). In some embodiments, the cutoff 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% of cell-free DNA or a combination of cell-free DNA and RNA. In some embodiments, the cutoff threshold is adjusted depending on the type of organ being transplanted. In some embodiments, the cutoff threshold is adjusted depending on the number of organs being transplanted.

[0177] In some embodiments, the cutoff threshold is an estimated percentage of donor-derived RNA relative to total RNA or a function thereof. In some embodiments, the cutoff 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 cutoff 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 cutoff threshold is adjusted depending on the type of organ being transplanted. In some embodiments, the cutoff threshold is adjusted depending on the number of organs being transplanted.

[0178] In some embodiments, the cutoff threshold is an estimated percentage of donor-derived mRNA relative to total mRNA, or a function thereof. In some embodiments, the cutoff 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 cutoff 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 cutoff threshold is adjusted depending on the type of organ being transplanted. In some embodiments, the cutoff threshold is adjusted depending on the number of organs being transplanted.

[0179] In some embodiments, the cutoff threshold is an estimated percentage of donor-derived preselected mRNA targets relative to total mRNA, or a function thereof. In some embodiments, the cutoff 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 cutoff 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 preselected mRNA targets. In some embodiments, the cutoff threshold is adjusted depending on the type of organ being transplanted. In some embodiments, the cutoff threshold is adjusted depending on the number of organs being transplanted.

[0180] In some embodiments, the cutoff threshold is an estimated percentage of donor-derived protein to total protein or a function thereof. In some embodiments, the cutoff 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 cutoff 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% of cell-free DNA or the combination of cell-free DNA and protein. In some embodiments, the cutoff threshold is adjusted depending on the type of organ being transplanted. In some embodiments, the cutoff threshold is adjusted depending on the number of organs being transplanted.

[0181] In some embodiments, the cutoff threshold is proportional to the absolute value of the donor-derived RNA concentration. In some embodiments, the cutoff threshold is the copy number of donor-derived RNA or a function thereof. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of RNA. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of RNA per volume unit of blood sample. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of RNA per volume unit of blood sample multiplied by the transplant recipient's body weight, BMI, or blood volume.

[0182] In some embodiments, the cutoff threshold is proportional to the absolute value of the donor-derived RNA concentration. In some embodiments, the cutoff threshold is the copy number of donor-derived RNA or a function thereof. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of RNA. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of RNA per volume unit of blood sample. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of RNA per volume unit of blood sample multiplied by the transplant recipient's body weight, BMI, or blood volume.

[0183] In some embodiments, the cutoff threshold is proportional to the absolute value of the concentration of the donor-derived protein. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of protein. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of protein per volume unit of blood sample. In some embodiments, the cutoff threshold is expressed as the amount or absolute amount of protein per volume unit of blood sample multiplied by the body weight, BMI, or blood volume of the transplant recipient.

[0184] In some embodiments, the method further comprises repeating steps (a)-(d) longitudinally on the same transplant recipient and determining longitudinal changes in the amount of RNA or a function thereof and the amount of donor-derived RNA or a function thereof. In some embodiments, the method further comprises repeating steps (a)-(d) longitudinally on the same transplant recipient and determining longitudinal changes in the amount of protein or a function thereof and the amount of donor-derived protein or a function thereof.

[0185] definition As used herein, the term "single nucleotide polymorphism (SNP)" refers to a single nucleotide that may differ between the genomes of two members of the same species. The use of this term does not imply any restriction on the frequency with which each variant occurs.

[0186] In some embodiments, for example, sequence refers to a DNA or RNA sequence or a gene sequence. It may refer to the primary physical structure of a DNA or RNA molecule or strand in an individual. It may refer to the sequence of nucleotides present in that DNA or RNA molecule, or the complementary strand of a DNA or RNA molecule. It may refer to the information contained in a DNA or RNA molecule as its in silico representation.

[0187] In some embodiments, for example, a locus refers to a particular region of interest on an individual's DNA or RNA, including, but not limited to, one or more SNPs, potential insertion or deletion sites, or sites of some other associated genetic variation. A disease-associated SNP can also refer to a disease-associated locus.

[0188] In some embodiments, for example, a polymorphic allele, or "polymorphic locus," refers to an allele or locus whose genotype varies between individuals within a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms (SNPs), short tandem repeats, deletions, duplications, and inversions.

[0189] In some embodiments, for example, an allele refers to a nucleotide or nucleotide sequence that occupies a particular locus.

[0190] In some embodiments, for example, genotype data, or "genotype data," refers to data describing aspects of one or more individual's genome. It can refer to one or a set of loci, a partial or entire sequence, a partial or entire chromosome, or the entire genome. It can refer to the identity of one or more nucleotides. It can refer to a set of contiguous nucleotides, or nucleotides at different locations in the genome, or a combination thereof. While genotype data is computational, it is also possible to think of the physical nucleotides in a sequence as chemically encoded genetic data. Genotype data can be referred to as "pertaining to" an individual(s), "of" an individual(s), "at" an individual(s), "from" an individual(s), or "relating to" an individual. Genotype data can refer to output measurements from a genotyping platform where these measurements are made on genetic material.

[0191] In some embodiments, for example, genetic material, or "genetic sample," refers to physical matter, such as tissue or blood, from one or more individuals that contains nucleic acid (including, for example, DNA or RNA).

[0192] In some embodiments, for example, allele data refers to a set of genotype data for a set of one or more alleles. It can refer to graded haplotype data. It can refer to SNP identities, and it can refer to nucleic acid sequence data, including insertions, deletions, repeats, and mutations.

[0193] In some embodiments, for example, an allele state refers to the actual state of a gene within a set of one or more alleles. This can refer to the actual state of a gene as described by allele data.

[0194] In some embodiments, for example, allele ratio or allele ratio refers to the ratio between the amount of each allele at a locus present in a sample or individual. If the sample is measured by sequencing, the allele ratio may refer to the proportion of sequencing reads that map to each allele at the locus. If the sample is measured by an intensity-based measurement method, the allele ratio may refer to the proportion of the amount of each allele present at that locus as estimated by the measurement method.

[0195] In some embodiments, for example, the allele count refers to the number of sequences that map to a particular locus, or, if the locus is polymorphic, the number of sequences that map to each of the alleles. If each allele is counted in a binary manner, the allele count will be an integer. If alleles are counted probabilistically, the allele count may be a fraction.

[0196] In some embodiments, for example, a primer, or a "PCR probe," refers to a single DNA molecule (DNA oligomer) or a collection of DNA molecules (DNA oligomers) where the DNA molecules are identical or nearly identical, the primer includes a region designed to hybridize to a target polymorphic locus, and includes a priming sequence designed to enable amplification, such as PCR amplification. A primer may also include a molecular barcode. A primer may include a random region that is different for each individual molecule.

[0197] In some embodiments, for example, a hybrid capture probe refers to any nucleic acid sequence, possibly modified, generated by various methods, such as PCR or direct synthesis, that is intended to be complementary to one strand of a specific target DNA or RNA sequence in a sample. An exogenous hybrid capture probe may be added to a prepared sample and hybridized through a denaturation-annealing process to form exogenous-endogenous fragment duplexes. These duplexes can then be physically separated from the sample by various means.

[0198] In some embodiments, for example, a sequencing read refers to data representing a sequence of nucleotide bases measured using a clonal sequencing method. Clonal sequencing can generate sequence data representing a single, clone, or cluster of original DNA or RNA molecules. A sequencing read can also have an associated quality score for each base position in the sequence, indicating the likelihood that the nucleotide was called correctly.

[0199] In some embodiments, for example, mapping a sequencing read is the process of determining the location of the origin of a sequencing read in the genomic sequence of a particular organism. The location of the origin of a sequencing read is based on the similarity of the nucleotide sequences of the read and the genomic sequence.

[0200] In some embodiments, for example, DNA or RNA of donor origin refers to DNA or RNA that was originally part of cells whose genotype was essentially equivalent to that of the transplant donor. The donor can be a human or a non-human mammal (e.g., a pig).

[0201] In some embodiments, for example, DNA or RNA of recipient origin refers to DNA or RNA that was originally part of a cell whose genotype was essentially equivalent to that of the transplant recipient.

[0202] In some embodiments, RNA may refer to messenger RNA (mRNA), small non-coding RNA (sncRNA), transfer RNA (tRNA), or non-protein-coding RNA from a cell. In some embodiments, sncRNA includes microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), or other RNA (miscRNA). In some embodiments, the RNA is cell-free RNA. In some embodiments, the cell-free RNA is derived from exosomes or microvesicles.

[0203] In some embodiments, amplification of RNA involves reverse transcribing the RNA to produce complementary DNA (cDNA), followed by amplification of the cDNA by amplification methods disclosed elsewhere herein.

[0204] In some embodiments, for example, transplant recipient plasma refers to the plasma portion from blood from a female patient who has received an allograft or xenograft, eg, an organ transplant recipient.

[0205] In some embodiments, for example, preferential enrichment of DNA or RNA corresponding to a locus, or preferential enrichment of DNA or RNA at a locus, refers to any technique that results in a higher proportion of DNA or RNA molecules in a post-enrichment DNA or RNA mixture corresponding to a locus than the proportion of DNA or RNA molecules in the pre-enrichment DNA or RNA mixture corresponding to the locus. The technique may include selective amplification of DNA or RNA molecules corresponding to the locus. The technique may include removing DNA or RNA molecules that do not correspond to the locus. The technique may include a combination of methods. Enrichment is defined as the proportion of DNA or RNA molecules in the post-enrichment mixture corresponding to the locus divided by the proportion of DNA or RNA molecules in the pre-enrichment mixture corresponding to the locus. Preferential enrichment may be performed at multiple loci. In some embodiments of the present disclosure, the enrichment is greater than 20. In some embodiments of the present disclosure, the enrichment is greater than 200. In some embodiments of the present disclosure, the enrichment is greater than 2,000. When preferential enrichment is performed at multiple loci, enrichment may refer to the average enrichment of all loci in the set of loci.

[0206] In some embodiments, for example, amplification refers to techniques that increase the copy number of RNA and / or DNA molecules.

[0207] In some embodiments, for example, selective amplification can refer to techniques that increase the copy number of specific RNA and / or DNA molecules or RNA and / or DNA molecules corresponding to specific regions of RNA and / or DNA. It can also refer to techniques that increase the copy number of specific target RNA and / or DNA molecules or target regions of RNA and / or DNA over non-target RNA and / or DNA molecules or regions. Selective amplification can be a method of preferential enrichment.

[0208] In some embodiments, for example, a universal priming sequence refers to a DNA sequence that can be added to a population of target nucleic acid molecules by, for example, ligation, PCR, or ligation-mediated PCR. Once added to a population of target molecules, primers specific to the universal priming sequence can be used to amplify the target population using a single amplification primer pair. The universal priming sequence does not need to be related to the target sequence.

[0209] In some embodiments, for example, a universal adaptor, or "ligation adaptor" or "library tag," is a DNA molecule that contains universal priming sequences that can be covalently attached to the 5' and 3' ends of a population of target double-stranded DNA molecules. The addition of the adaptor provides universal priming sequences at the 5' and 3' ends of the target population from which PCR amplification can be performed, amplifying all molecules from the target population using a single amplification primer pair.

[0210] In some embodiments, for example, targeting refers to a method used to selectively amplify or otherwise preferentially enrich DNA or RNA molecules corresponding to a set of loci in a mixture of DNA or RNA.

[0211] Analysis of donor-derived RNA to monitor xenograft rejection "Acute rejection (AR)" is a rejection reaction by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by the recipient's immune cells infiltrating the transplanted tissue and exerting effector functions to destroy the transplanted tissue. The onset of acute rejection is rapid and typically occurs in humans within a few weeks of transplant surgery. Generally, acute rejection can be inhibited or suppressed by immunosuppressive drugs such as rapamycin, cyclosporin A, and anti-CD40L monoclonal antibodies.

[0212] "Chronic transplant rejection or injury" or "CAI" typically occurs in humans within months to years after engraftment, even when immunosuppression of acute rejection is successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can usually be described by a series of specific disorders characteristic of a particular organ. For example, in lung transplants, such disorders include fibroproliferative destruction of the airways (bronchiolitis obliterans); in cardiac tissue transplants, such as heart transplants or valve replacements, such disorders include fibroatherosclerosis; in kidney transplants, such disorders include obstructive nephropathy, nephrosclerosis, and tubulointerstitial nephropathy; and in liver transplants, such disorders include vanishing bile duct syndrome. Chronic rejection is also characterized by ischemic injury, denervation of the transplanted tissue, and immunosuppressant-related hyperlipidemia and hypertension.

[0213] The term "xenograft rejection" includes both acute and chronic transplant rejection. The term xenograft refers to transplants in which the recipient is a different species from the donor. In particular, the recipient may be human and the donor may be porcine. The term "graft injury" refers to all modes of graft dysfunction, regardless of pathological diagnosis. The term "organ injury" refers to biomarkers that track organ functional decline, whether the organ is native or transplanted, and regardless of etiology.

[0214] In some embodiments, the method further comprises performing universal amplification of the extracted RNA, hi some embodiments, the universal amplification preferentially amplifies RNA from the donor over RNA from the recipient.

[0215] In some embodiments, the transplant recipient is a mammal, hi some embodiments, the transplant recipient is a human.

[0216] In some embodiments, the transplant recipient is receiving a transplant selected from an organ transplant, a tissue transplant, a cell transplant, and a bodily fluid transplant. In some embodiments, the transplant recipient is receiving a transplant selected from a kidney transplant, a liver transplant, a pancreas transplant, an intestinal transplant, a heart transplant, a lung transplant, a heart / lung transplant, a stomach transplant, a testis transplant, a penis transplant, an ovary transplant, a uterus transplant, a thymus transplant, a face transplant, a hand transplant, a leg transplant, a bone transplant, a bone marrow transplant, a cornea transplant, a skin transplant, a pancreatic islet cell transplant, a heart valve transplant, a blood vessel transplant, and a blood transfusion. In some embodiments, the transplant recipient is receiving a SPK transplant.

[0217] In some embodiments, the method further comprises using the quantified amount of donor-derived RNA to detect the occurrence or likelihood of occurrence of active rejection of the transplant, hi some embodiments, the method is performed without prior knowledge of the donor genotype.

[0218] In some embodiments, each primer pair is designed to amplify a target sequence of approximately 50-100 bp. In some embodiments, each primer pair is designed to amplify a target sequence of 75 bp or less. In some embodiments, each primer pair is designed to amplify a target sequence of approximately 60-75 bp. In some embodiments, each primer pair is designed to amplify a target sequence of approximately 65 bp.

[0219] In some embodiments, the target amplification comprises amplifying at least 1,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 2,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 5,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 10,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying about 100 to about 50,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying about 1,000 to about 50,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying about 5,000 to about 50,000 polymorphic loci in a single reaction volume.

[0220] In some embodiments, the method further comprises measuring the abundance of one or more alleles at a target locus that is a polymorphic locus. In some embodiments, the polymorphic locus and the non-polymorphic locus are amplified in a single reaction.

[0221] In some embodiments, the quantifying step comprises detecting the amplified target loci using a microarray. In some embodiments, the quantifying step does not comprise using a microarray.

[0222] In some embodiments, target amplification involves simultaneously amplifying 50-50,000 target loci in a single reaction volume using (i) at least 50-50,000 different primer pairs, or (ii) at least 50-50,000 target-specific primers and 50-50,000 primer pairs of universal or tag-specific primers.

[0223] 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 of 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 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 higher amount of dd-RNA indicates a higher likelihood of transplant rejection.

[0224] In a further aspect, the present invention relates to a method for diagnosing acute xenograft rejection in a xenograft recipient, the method comprising: extracting RNA from a blood sample of the xenograft recipient, wherein the RNA includes donor-derived RNA and recipient-derived RNA; performing universal amplification of the extracted RNA; performing targeted amplification at 50-50,000 target loci in a single reaction volume using 50-50,000 primer pairs, wherein the target loci include polymorphic 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 acute xenograft rejection.

[0225] In some embodiments, the xenograft rejection is antibody-mediated transplant rejection. In some embodiments, the transplant rejection is T-cell mediated transplant rejection.

[0226] 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 xenograft undergoes borderline rejection, other damage, or is stable.

[0227] In a further aspect, the present invention relates to a method of monitoring immunosuppressive therapy in a subject, the method comprising: extracting RNA from a blood sample of a xenotransplant recipient, wherein the RNA comprises donor-derived RNA and recipient-derived RNA; performing universal amplification of the extracted RNA; 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 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 level of dd-RNA over a time interval indicates xenotransplant status.

[0228] In some embodiments, the method further comprises adjusting immunosuppressive therapy based on the level of dd-RNA over a time interval.

[0229] In some embodiments, an increase in the level of dd-RNA indicates transplant rejection and the need for adjustment of immunosuppressive therapy, while in some embodiments, an unchanged or decreased level of dd-RNA indicates transplant tolerance or stability and the need for adjustment of immunosuppressive therapy.

[0230] 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.

[0231] In one aspect, the disclosure relates to a method of administering 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; (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 dosage of immunosuppressive therapy according to, or a function of, the amount of cell-free DNA and the amount of donor-derived cell-free DNA.

[0232] In some embodiments, the amount of donor-derived cell-free DNA is measured by extracting cell-free DNA from a blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted cell-free DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification of the extracted DNA at 200 to 50,000 target loci in a single reaction volume; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of donor-derived cell-free DNA based on the sequencing reads.

[0233] In some embodiments, the method further comprises repeating steps (a)-(b) longitudinally 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.

[0234] In some embodiments, the methods disclosed herein further comprise titrating the dosage of 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.

[0235] In one aspect, the disclosure relates to a method of administering 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 the blood, plasma, serum, or urine sample of the transplant recipient; and (c) titrating the dosage of immunosuppressive therapy according to, or a function of, the amount of cell-free DNA and the amount of donor-derived RNA.

[0236] In some embodiments, the amount of donor-derived RNA is measured by extracting RNA from a blood, plasma, serum, or urine sample of the transplant recipient, wherein the extracted RNA includes donor-derived RNA and recipient-derived RNA; preparing a composition of amplified complementary DNA (cDNA) derived from the extracted RNA by performing multiplexed targeted amplification of cDNA at 200 to 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.

[0237] In some embodiments, the methods herein further include repeating steps (a)-(b) longitudinally on the same transplant recipient and determining longitudinal changes in the amount of RNA or a function thereof and longitudinal changes in the amount of donor-derived RNA or a function thereof.

[0238] In some embodiments, the methods disclosed herein further comprise titrating the dosage of immunosuppressive therapy according to 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.

[0239] 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 will undergo borderline rejection, other damage, or will be stable.

[0240] In some embodiments, the method does not include determining the genotype of the transplant donor and / or transplant recipient.

[0241] In some embodiments, the method further comprises measuring the abundance of one or more alleles at a target locus that is a polymorphic locus.

[0242] In some embodiments, the xenograft recipient is a human. In some embodiments, the xenograft recipient is receiving a xenograft selected from a kidney transplant, a liver transplant, a pancreas transplant, a pancreatic 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 is receiving a SPK transplant.

[0243] In some embodiments, the extraction step includes size selection to enrich for donor-derived RNA and reduce the amount of recipient-derived RNA that is discarded from ruptured white blood cells.

[0244] In some embodiments, the universal amplification step preferentially amplifies donor-derived RNA over recipient-derived RNA that is discarded from ruptured white blood cells.

[0245] In some embodiments, the method includes longitudinally collecting multiple blood samples from the xenotransplant recipient after transplantation and repeating steps (a)-(e) for each collected blood sample. In some embodiments, the method includes collecting and analyzing blood samples from the xenotransplant recipient over a period of about 3 months, about 6 months, about 12 months, about 18 months, or about 24 months, etc. In some embodiments, the method includes collecting blood samples from the xenotransplant recipient at intervals of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, or about 3 months, etc.

[0246] In some embodiments, the method has a sensitivity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in identifying acute rejection (AR) over non-AR, at a cutoff threshold of 1% dd-RNA and a 95% confidence interval.

[0247] In some embodiments, the method has a specificity of at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% in identifying AR over non-AR at a cutoff threshold of 1% dd-RNA and a 95% confidence interval.

[0248] 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 over non-AR at a cutoff threshold of 1% dd-RNA and a 95% confidence interval.

[0249] In some embodiments, the method has a sensitivity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in identifying AR over normal stable allografts (STA) at a cutoff threshold of 1% dd-RNA and a 95% confidence interval.

[0250] In some embodiments, the method has a specificity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in identifying AR over STA at a cutoff threshold of 1% dd-RNA and a 95% confidence interval.

[0251] 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 over STA at a cutoff threshold of 1% dd-RNA and a 95% confidence interval.

[0252] In some embodiments, the method has a sensitivity determined by a limit of blank (LoB) of 0.5% or less and a limit of detection (LoD) of 0.5% or less. In some embodiments, the LoB is 0.23% or less and the LoD is 0.29% or less. In some embodiments, the sensitivity is further determined by the limit of quantitation (LoQ). In some embodiments, the LoQ may be 10-fold greater than the LoD, the LoQ may be 5-fold greater than the LoD, the LoQ may be 1.5-fold greater than the LoD, the LoQ may be 1.2-fold greater than the LoD, the LoQ may be 1.1-fold greater than the LoD, or the LoQ may be equal to or greater than the LoD. In some embodiments, the LoB is 0.04% or less, the LoD is 0.05% or less, and / or the LoQ is equal to the LoD.

[0253] 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 value, and the R value is about 0.98 to about 1.0. In some embodiments, the R 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 fraction as a function of the corresponding attempted spike level, where the slope value is about 0.9 to about 1.2 and the intercept value is about -0.0001 to about 0.01. In some embodiments, the slope value is about 1 and the intercept value is about 0.

[0254] In some embodiments, the method has a precision determined by calculating the coefficient of variation (CV), wherein the CV is less than about 10.0%. The CV is less than about 6%. In some embodiments, the CV is less than about 4%. In some embodiments, the CV is less than about 2%. In some embodiments, the CV is less than about 1%.

[0255] In some embodiments, the AR is antibody-mediated rejection (ABMR). In some embodiments, the AR is T-cell-mediated rejection (TCMR).

[0256] Further disclosed herein are methods for detecting xenograft donor-derived RNA (dd-RNA) in a sample from a xenograft recipient. In some embodiments, in the methods disclosed herein, the xenograft recipient is a mammal. In some embodiments, the xenograft recipient is a human. In some embodiments, the xenograft is derived from a pig, a primate, a baboon, a cow, or a dog.

[0257] In some embodiments, the transplant recipient is undergoing a xenograft selected from a kidney transplant, a liver transplant, a pancreas transplant, a pancreatic 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 is undergoing a SPK transplant. In some embodiments, the method can be performed on the transplant recipient on the day of the transplant surgery, or after the transplant surgery, up to one year after the transplant surgery.

[0258] In some embodiments, disclosed herein are methods for amplifying target loci in donor-derived RNA (dd-RNA) from a blood sample of a transplant recipient, the method comprising: a) extracting RNA from the 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 target loci, wherein the target loci comprise 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; and c) amplifying the target loci.

[0259] In some embodiments, disclosed herein are methods for detecting donor-derived RNA (dd-RNA) in a sample from a transplant recipient, the method comprising: (a) extracting RNA from a sample from the transplant recipient, the RNA including RNA from both the transplanted cells and the transplant recipient; (b) enriching the extracted RNA at target loci, the target loci including 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; (c) amplifying the target loci; (d) contacting the amplified target loci with probes that specifically hybridize to the target loci; and (e) detecting binding of the probes to the target loci, thereby detecting the RNA in the blood sample. In some embodiments, the probes are labeled with a detectable marker.

[0260] In some embodiments, disclosed herein are methods for determining the likelihood of transplant rejection in a transplant recipient, the method comprising: a) extracting RNA from a sample of the transplant recipient, wherein the RNA includes RNA from both the transplanted cells and the transplant recipient; b) enriching the extracted RNA at target loci, wherein the target loci include 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplant RNA and the amount of recipient RNA in the recipient sample, wherein a higher amount of dd-RNA indicates a higher likelihood of transplant rejection.

[0261] In some embodiments, provided herein are methods for diagnosing acute transplant rejection in a transplant recipient, the method comprising: a) extracting RNA from a blood sample of the transplant recipient, wherein the RNA includes RNA from both the transplanted cells and the transplant recipient; b) enriching the extracted RNA at target loci, wherein the target loci include 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplant 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 acute transplant rejection.

[0262] 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 will undergo borderline rejection, other damage, or will be stable.

[0263] In some embodiments, disclosed herein are methods for monitoring immunosuppressive therapy in a subject, the methods comprising: a) extracting RNA from a blood sample of a transplant recipient, the RNA including RNA from both the transplanted cells and the xenograft recipient; b) enriching the extracted RNA at target loci, the target loci including 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of xenograft DNA and the amount of recipient DNA in the recipient blood sample, wherein a change in the level of dd-RNA over a time interval indicates transplant status. In some embodiments, the method further comprises adjusting the immunosuppressive therapy based on the level of dd-RNA over a time interval. In some embodiments, an increase in the level of dd-RNA indicates transplant rejection and the need for adjustment of immunosuppressive therapy. In some embodiments, a change or decrease in the level of dd-RNA indicates xenograft tolerance or stability and the need for adjustment of immunosuppressive therapy.

[0264] In some embodiments, in the methods disclosed herein, the target locus is amplified with an amplicon about 50-100 bp in length, or about 60-80 bp in length, in some embodiments, the amplicon is about 65 bp in length.

[0265] In some embodiments, the methods disclosed herein further comprise measuring the amount of transplanted RNA and the amount of recipient RNA in a recipient blood sample.

[0266] In some embodiments, the methods disclosed herein do not include determining the genotype of the xenograft donor and the xenograft recipient.

[0267] In some embodiments, the methods disclosed herein further comprise detecting the amplified target loci using a microarray.

[0268] In some embodiments, in the methods disclosed herein, polymorphic loci and non-polymorphic loci are amplified in a single reaction.

[0269] In some embodiments, in the methods disclosed herein, RNA is preferentially enriched at a target locus.

[0270] In some embodiments, preferentially enriching RNA in a sample at a plurality of target loci includes obtaining a plurality of pre-circularized probes, each probe targeted to one of the target loci, and the 3' and 5' ends of the probes designed to hybridize to a region of RNA separated from a polymorphic site at the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the pre-circularized probes to RNA converted to cDNA from the sample; filling in gaps between the ends of the hybridized probes using a DNA polymerase; circularizing the pre-circularized probes; and amplifying the circularized probes.

[0271] In some embodiments, preferentially enriching RNA at a plurality of polymorphic loci includes obtaining a plurality of ligation-mediated PCR probes, each PCR probe targeting one of the polymorphic loci, and upstream and downstream PCR probes designed to hybridize to cDNA regions obtained from the extracted RNA on one strand of the cDNA that are separated from the polymorphic site of the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the ligation-mediated PCR probes to the cDNA from the first sample; using DNA polymerase to fill gaps between the ends of the ligation-mediated PCR probes; ligating the ligation-mediated PCR probes; and amplifying the ligated ligation-mediated PCR probes.

[0272] In some embodiments, preferentially enriching RNA at a plurality of target loci includes obtaining a plurality of hybrid capture probes that target specific loci, hybridizing the hybrid capture probes to RNA in the sample, and physically removing some or all of the unhybridized RNA from the first RNA sample.

[0273] In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping. In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping, and the length of the capture probes on either side can be selected from the group consisting of less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases. In some embodiments, the hybrid capture probes are designed to hybridize to regions that overlap the polymorphic site, and the plurality of hybrid capture probes includes at least two hybrid capture probes for each polymorphic locus, each hybrid capture probe designed to be complementary to a different allele at the polymorphic locus.

[0274] In some embodiments, preferentially enriching RNA or RNA-derived cDNA at a plurality of polymorphic loci includes obtaining a plurality of inner forward primers, each primer targeting one of the polymorphic loci, the 3' end of the inner forward primer designed to hybridize to a region of the RNA or RNA-derived cDNA upstream of the polymorphic site and separated from the polymorphic site by a small number of bases, wherein the small number of bases is selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, or 31-60 base pairs; and optionally, each primer is a polymorphic locus. obtaining a plurality of inner reverse primers that target one of the polymorphic loci, the inner reverse primers being designed such that their 3' ends hybridize to a region of the RNA or RNA-derived cDNA upstream of the polymorphic site and separated from the polymorphic site by a small number of bases, where the small number of bases is selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, or 31-60 base pairs; hybridizing the inner primers to the RNA or RNA-derived cDNA; and amplifying the cDNA using polymerase chain reaction to form an amplicon.

[0275] In some embodiments, the method also includes obtaining a plurality of outer forward primers, each targeting one of the polymorphic loci and designed to hybridize to a region of the RNA or RNA-derived cDNA upstream of the inner forward primer; optionally obtaining a plurality of outer reverse primers, each targeting one of the polymorphic loci and designed to hybridize to a region of the RNA or RNA-derived cDNA immediately downstream of the inner reverse primer; hybridizing the first primer to the RNA or RNA-derived cDNA; and amplifying the cDNA using polymerase chain reaction.

[0276] In some embodiments, the method also includes obtaining a plurality of outer reverse primers, each targeting one of the polymorphic loci and designed to hybridize to a region of the RNA or RNA-derived cDNA immediately downstream of the inner reverse primer; optionally obtaining a plurality of outer forward primers, each targeting one of the polymorphic loci and designed to hybridize to a region of the RNA or RNA-derived cDNA upstream of the inner forward primer; hybridizing a first primer to the RNA or RNA-derived cDNA; and amplifying the RNA or RNA-derived cDNA using polymerase chain reaction.

[0277] In some embodiments, preparing the first sample further comprises adding universal adaptors 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 are less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp, which portion is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%.

[0278] In some embodiments, amplifying RNA or RNA-derived cDNA is carried out in one or more separate reaction volumes, each separate reaction volume comprising 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.

[0279] In some embodiments, preparing the sample further comprises dividing the sample into multiple portions, and the RNA or RNA-derived cDNA of each portion is preferentially enriched for a subset of the multiple polymorphic loci. In some embodiments, the inner primers are selected by identifying primer pairs that are likely to form undesired primer duplexes and removing at least one of the identified primer pairs that are likely to form undesired primer duplexes from the multiple primers. In some embodiments, the inner primers include a region designed to hybridize either upstream or downstream of the targeted polymorphic locus, and optionally include a universal priming sequence designed to enable PCR amplification. In some embodiments, at least some of the primers further include a random region that varies for each individual primer molecule. In some embodiments, at least some of the primers further include a molecular barcode.

[0280] In some embodiments, the method includes (a) performing a multiplex polymerase chain reaction (PCR) on a nucleic acid sample containing target loci to simultaneously amplify at least 1,000 different target loci in a single reaction volume using (i) at least 1,000 different primer pairs, or (ii) at least 1,000 target-specific primers and either a universal primer or a tag-specific primer, to generate amplification products containing target amplicons, and (b) sequencing the amplified products. In some embodiments, the method does not include using a microarray.

[0281] In some embodiments, the method includes (a) performing a multiplex polymerase chain reaction (PCR) on an RNA (or cDNA) sample containing target loci to simultaneously amplify at least 1,000 different target loci in a single reaction volume using (i) at least 1,000 different primer pairs, or (ii) at least 1,000 target-specific primers and either a universal primer or a tag-specific primer, to generate amplification products containing target amplicons, and b) sequencing the amplified products. In some embodiments, the method does not include using a microarray.

[0282] In some embodiments, the method also includes obtaining genotype data from one or both of the transplant donor and the transplant recipient. In some embodiments, obtaining genotype data from one or both of the transplant donor and the transplant recipient includes preparing RNA from the donor and the recipient, the preparation including preferentially enriching the RNA at the plurality of polymorphic loci to obtain an RNA or cDNA preparation, optionally amplifying the prepared cDNA, and measuring the RNA in the prepared sample at the plurality of polymorphic loci.

[0283] In some embodiments, constructing a joint distribution model of expected allele count probabilities for multiple polymorphic loci on a chromosome is performed using genotype data obtained from one or both of the transplant donor and the transplant recipient. In some embodiments, the first sample is isolated from the plasma of the transplant recipient, and obtaining the genotype data from the transplant recipient is performed by estimating the recipient's genotype data from RNA measurements performed on the prepared sample.

[0284] In some embodiments, the preferential enrichment results in an average degree of allelic bias between the prepared sample and the first sample of a factor selected from the group consisting of 2-fold or less, 1.5-fold or less, 1.2-fold or less, 1.1-fold or less, 1.05-fold or less, 1.02-fold or less, 1.01-fold or less, 1.005-fold or less, 1.002-fold or less, 1.001-fold or less, and 1.0001-fold or less. In some embodiments, the plurality of polymorphic loci are SNPs. In some embodiments, measuring RNA in the prepared sample is performed by sequencing.

[0285] In some embodiments, a diagnostic box is disclosed to aid in determining the transplant status of a transplant recipient, where the diagnostic box can perform the preparation and measurement steps of the disclosed methods.

[0286] In some embodiments, the allele counts are probabilistic rather than binary. In some embodiments, measurements of RNA from samples prepared at multiple polymorphic loci are also used to determine whether a transplant has inherited one or more linked haplotypes.

[0287] In some embodiments, constructing a joint distribution model of allele count probabilities is done by modeling the dependency between polymorphic alleles on a chromosome using data on the probability of chromosome crossover at different locations on the chromosome. In some embodiments, constructing a joint distribution model of allele counts and determining the relative probability of each hypothesis is done using a method that does not require the use of a reference chromosome.

[0288] In some embodiments, determining the relative probability of each hypothesis utilizes estimated proportions of donor-derived RNA (dd-RNA) in the prepared sample. In some embodiments, the DNA measurements from the prepared sample used to calculate allele count probabilities and determine the relative probability of each hypothesis include primary genotype data. In some embodiments, selecting the transplant state corresponding to the hypothesis with the greatest probability is performed using maximum likelihood or maximum a posteriori estimates.

[0289] In some embodiments, calling the transplantation status also includes combining the relative probability and allele count probability of each status hypothesis determined using the joint distribution model with the relative probability of each status hypothesis calculated using statistical methods derived from the group consisting of read count analysis, heterozygosity rate comparisons, statistics available only using donor genetic information, normalized genotype signal probabilities for a particular donor / recipient context, statistics calculated using estimated transplantation fractions for the first sample or prepared sample, and combinations thereof.

[0290] In some embodiments, a reliability estimate is calculated for the called transplant status. In some embodiments, the method also includes taking a clinical action based on the called transplant status.

[0291] In some embodiments, a report indicating the determined transplant status is generated using this method. In some embodiments, a kit for determining transplant status designed for use with the methods disclosed herein is disclosed, the kit including a plurality of inner forward primers and optionally a plurality of inner reverse primers, each primer designed to hybridize to a region of RNA immediately upstream and / or downstream from one of the target sites, wherein the hybridization region is separated from the polymorphic site by a small number of bases, the small number selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-60, and combinations thereof.

[0292] In some embodiments, the methods disclosed herein include a selection step to select for shorter RNAs.

[0293] In some embodiments, the methods disclosed herein include a universal application step for concentrating RNA.

[0294] In some embodiments, a determination that the amount of dd-RNA is above a cutoff threshold indicates acute rejection of the transplant. Machine learning and artificial intelligence may be used to determine rejection and non-rejection.

[0295] In some embodiments, the cutoff threshold is expressed as the percentage of dd-RNA in the blood sample (dd-RNA%).

[0296] In some embodiments, the cutoff threshold is expressed as the number of copies of dd-RNA per unit volume of blood sample.

[0297] In some embodiments, the cutoff threshold is expressed as the number of copies of dd-RNA per unit volume of blood sample multiplied by the body weight or blood volume of the transplant recipient.

[0298] In some embodiments, the cutoff threshold takes into account the patient's weight or blood volume.

[0299] In some embodiments, the cutoff threshold takes into account one or more of donor genome copies per volume of plasma, cell-free DNA or RNA yield per volume of plasma, donor height, donor weight, donor age, donor sex, donor ethnicity, donor organ weight, donor organ, living vs. deceased donor, related vs. unrelated donor, recipient height, recipient weight, recipient age, recipient sex, recipient ethnicity, creatinine, eGFR (estimated glomerular filtration rate), cfDNA methylation, DSA (donor-specific antibodies), KDPI (Kidney Donor Profile Index), medications (immunosuppressants, steroids, anticoagulants, etc.), infections (BKV, EBV, CMV, UTI), recipient and / or donor HLA allele or epitope mismatch, Banff classification of renal allograft pathology, and cause-specific vs. surveillance or protocol biopsy.

[0300] In some embodiments, the cutoff threshold is scaled according to the amount of total RNA in the blood sample.

[0301] In some embodiments, the method has a sensitivity of at least 80% with a confidence interval of 95% in identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in the sample.

[0302] In some embodiments, the method has a specificity of at least 70% with a confidence interval of 95% in identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in the blood sample.

[0303] In some embodiments, the method has a sensitivity of at least 80% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in the sample. In some embodiments, the method has a sensitivity of at least 85% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in a blood sample. In some embodiments, the method has a sensitivity of at least 90% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in a blood sample. In some embodiments, the method has a sensitivity of at least 95% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in the sample.

[0304] In some embodiments, this method has a specificity of at least 70% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of RNA is above a cutoff threshold scaled according to the amount of total RNA in a blood sample. In some embodiments, this method has a specificity of at least 75% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in a blood sample. In some embodiments, this method has a specificity of at least 85% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in a blood sample. In some embodiments, this method has a specificity of at least 90% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total RNA in a blood sample. In some embodiments, the method has at least 95% specificity, with a 95% confidence interval, in identifying acute rejection (AR) over non-AR when the amount of dd-RNA is above a cutoff threshold scaled according to the amount of total RNA in the blood sample.

[0305] Analysis of donor-derived cell-free DNA for monitoring xenograft rejection In one aspect, the present invention further comprises: (i) measuring the amount of donor-derived cell-free DNA in a sample obtained from the xenotransplant recipient and extracting cell-free DNA from the sample obtained from the xenotransplant recipient, wherein the extracted cell-free DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; (ii) performing targeted amplification of the extracted DNA at 10 to 50,000 target loci in a single reaction volume; (iii) sequencing the amplified DNA to obtain sequencing reads, quantifying the amount of donor-derived cell-free DNA based on the sequencing reads, and determining transplant rejection based on whether the amount of donor-derived cell-free DNA or a function thereof exceeds a cutoff threshold for the amount of cell-free DNA indicative of transplant rejection, wherein transplant rejection is determined based on whether both the amount of donor-derived RNA and the amount of donor-derived cell-free DNA, or a function thereof, exceed a cutoff threshold for indicating transplant rejection.

[0306] 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 the blood sample of the xenotransplant recipient, wherein the DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification at 100 to 50,000 or 500 to 50,000 target loci in a single reaction volume using 100 to 50,000 or 500 to 50,000 primer pairs, wherein the target loci include polymorphic loci and non-polymorphic loci, and each primer pair is designed to amplify a target sequence of 100 bp or less; and quantifying the amount of donor-derived cell-free DNA in the amplification products.

[0307] 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 the blood sample of the xenotransplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA, the extraction step comprising size selection to enrich the donor-derived cell-free DNA and reduce the amount of recipient-derived cell-free DNA discarded from ruptured white blood cells; 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 include polymorphic and non-polymorphic loci; and quantifying the amount of donor-derived cell-free DNA in the amplification products.

[0308] In another aspect, the 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 the blood sample of the transplant recipient, wherein the DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification at 100 to 50,000 target loci in a single reaction volume using 100 to 50,000 primer pairs, wherein the target loci comprise polymorphic and non-polymorphic loci; sequencing the amplification products by high-throughput sequencing; and quantifying the amount of donor-derived cell-free DNA.

[0309] In some embodiments, the method further comprises performing universal amplification of the extracted DNA, which in some embodiments preferentially amplifies donor-derived cell-free DNA over recipient-derived cell-free DNA discarded from ruptured white blood cells.

[0310] In some embodiments, the xenograft recipient is a mammal. In some embodiments, the transplant recipient is a human. In some embodiments, the xenograft is derived from a pig, a primate, a baboon, a cow, or a dog.

[0311] In some embodiments, the xenograft recipient is receiving a xenograft selected from an organ transplant, a tissue transplant, a cell transplant, and a bodily fluid transplant. In some embodiments, the transplant recipient is receiving a transplant selected from a kidney transplant, a liver transplant, a pancreas transplant, an intestinal transplant, a heart transplant, a lung transplant, a heart / lung transplant, a stomach transplant, a testis transplant, a penis transplant, an ovary transplant, a uterus transplant, a thymus transplant, a face transplant, a hand transplant, a leg transplant, a bone transplant, a bone marrow transplant, a cornea transplant, a skin transplant, a pancreatic islet cell transplant, a heart valve transplant, a blood vessel transplant, and a blood transfusion. In some embodiments, the transplant recipient is receiving a SPK transplant.

[0312] In some embodiments, the quantifying step comprises determining the ratio of donor-derived cell-free DNA to the sum of donor-derived cell-free DNA and recipient-derived cell-free DNA in the blood sample, hi some embodiments, the quantifying step comprises determining the number of copies of donor-derived cell-free DNA per unit volume of the blood sample.

[0313] In some embodiments, the method further comprises using the quantified amount of donor-derived cell-free DNA to detect the occurrence or likelihood of occurrence of active transplant rejection, hi some embodiments, the method is performed without prior knowledge of the donor's genotype.

[0314] In some embodiments, each primer pair is designed to amplify a target sequence of approximately 50-100 bp. In some embodiments, each primer pair is designed to amplify a target sequence of 75 bp or less. In some embodiments, each primer pair is designed to amplify a target sequence of approximately 60-75 bp. In some embodiments, each primer pair is designed to amplify a target sequence of approximately 65 bp.

[0315] In some embodiments, the target amplification comprises amplifying at least 1,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 2,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 5,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 10,000 polymorphic loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying 10 to 10,000, 10 to 50,000, 100 to 50,000, or 1000 to 50,000 polymorphic loci in a single reaction volume.

[0316] In some embodiments, the method further comprises measuring the abundance of one or more alleles at a target locus that is a polymorphic locus. In some embodiments, the polymorphic locus and the non-polymorphic locus are amplified in a single reaction.

[0317] In some embodiments, the quantifying step comprises detecting the amplified target loci using a microarray. In some embodiments, the quantifying step does not comprise using a microarray.

[0318] In some embodiments, target amplification involves simultaneously amplifying 500-50,000 target loci in a single reaction volume using (i) at least 500-50,000 different primer pairs, or (ii) at least 500-50,000 target-specific primers and 500-50,000 primer pairs of universal or tag-specific primers.

[0319] 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 of 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 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 higher amount of dd-cfDNA indicates a higher likelihood of transplant rejection.

[0320] In a further aspect, the present invention relates to a method for diagnosing acute rejection of a xenograft in a xenograft recipient, the method comprising: extracting DNA from a blood sample of the xenograft recipient, wherein the DNA includes donor-derived cell-free DNA and recipient-derived cell-free DNA; performing universal amplification of 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 include polymorphic 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 acute rejection of the xenograft.

[0321] In some embodiments, the xenograft rejection is antibody-mediated transplant rejection. In some embodiments, the xenograft rejection is T-cell mediated transplant rejection.

[0322] 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 will undergo borderline rejection, other damage, or will be stable.

[0323] 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 of 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 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 level of dd-cfDNA over a time interval is indicative of xenotransplant status.

[0324] In some embodiments, the method further comprises adjusting immunosuppressive therapy based on the level of dd-cfDNA over the time interval.

[0325] In some embodiments, an increased level of dd-cfDNA indicates xenograft rejection and the need for adjustment of immunosuppressive therapy. In some embodiments, an unchanged or decreased level of dd-cfDNA indicates xenograft tolerance or stability and the need for adjustment of immunosuppressive therapy.

[0326] 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 acute rejection of the xenograft. In some embodiments, the xenograft rejection is antibody-mediated transplant rejection. In some embodiments, the xenograft rejection is T-cell-mediated transplant rejection.

[0327] 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 will undergo borderline rejection, other damage, or will be stable.

[0328] In some embodiments, the method does not include determining the genotype of the transplant donor and / or transplant recipient.

[0329] In some embodiments, the method further comprises measuring the abundance of one or more alleles at a target locus that is a polymorphic locus.

[0330] In some embodiments, the target loci comprise 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.

[0331] In some embodiments, the target locus is amplified with an amplicon about 50-100 bp in length, or about 50-90 bp in length, or about 60-80 bp in length, or about 60-75 bp in length, or about 65 bp in length.

[0332] In some embodiments, the xenograft recipient is a human. In some embodiments, the transplant recipient is receiving a transplant selected from a kidney transplant, a liver transplant, a pancreas transplant, a pancreatic 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 is receiving a SPK transplant.

[0333] In some embodiments, the extraction step includes size selection to enrich for donor-derived cell-free DNA and reduce the amount of recipient-derived cell-free DNA that is discarded from ruptured white blood cells.

[0334] In some embodiments, the universal amplification step preferentially amplifies donor-derived cell-free DNA over recipient-derived cell-free DNA discarded from ruptured white blood cells.

[0335] In some embodiments, the method includes longitudinally collecting multiple blood samples from the transplant recipient after transplantation and repeating steps (a)-(e) for each collected blood sample. In some embodiments, the method includes collecting and analyzing blood samples from the transplant recipient over a period of about 3 months, or about 6 months, or about 12 months, or about 18 months, or about 24 months, etc. In some embodiments, the method includes collecting blood samples from the transplant recipient at intervals of about 1 week, or about 2 weeks, or about 3 weeks, or about 1 month, or about 2 months, or about 3 months, etc.

[0336] In some embodiments, the method has a sensitivity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in distinguishing acute rejection (AR) from non-AR at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0337] In some embodiments, the method has a specificity of at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% in distinguishing AR from non-AR at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0338] In some embodiments, the method has an area under the curve (AUC) of at least 0.8, or 0.85, or at least 0.9, or at least 0.95 in distinguishing AR from non-AR at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0339] In some embodiments, the method has a sensitivity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in distinguishing AR from normal stable allografts (STA) at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0340] In some embodiments, the method has a specificity of at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% in distinguishing AR over STA at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0341] In some embodiments, the method has an AUC of at least 0.8, or 0.85, or at least 0.9, or at least 0.95, or at least 0.98, or at least 0.99 in distinguishing AR over STA at a cutoff threshold of 1% dd-cfDNA and a 95% confidence interval.

[0342] In some embodiments, the method has a sensitivity determined by a limit of blank (LoB) of 0.5% or less and a limit of detection (LoD) of 0.5% or less. In some embodiments, the LoB is 0.23% or less and the LoD is 0.29% or less. In some embodiments, the sensitivity is further determined by the limit of quantitation (LoQ). In some embodiments, the LoQ may be 10-fold greater than the LoD, the LoQ may be 5-fold greater than the LoD, the LoQ may be 1.5-fold greater than the LoD, the LoQ may be 1.2-fold greater than the LoD, the LoQ may be 1.1-fold greater than the LoD, or the LoQ may be equal to or greater than the LoD. In some embodiments, the LoB is 0.04% or less, the LoD is 0.05% or less, and / or the LoQ is equal to the LoD.

[0343] In some embodiments, the method has an accuracy determined by evaluating a linear value obtained from a linear regression analysis of the measured donor fraction as a function of the corresponding attempted spike level, the linear value being R 2 is the value of R 2 In some embodiments, the value of R 2 The value is 0.999. In some embodiments, the method has an accuracy determined by calculating a slope value and an intercept value using linear regression for the measured donor fraction as a function of the corresponding attempted spike level, where the slope value is from about 0.9 to about 1.2 and the intercept value is from about -0.0001 to about 0.01. In some embodiments, the slope value is about 1 and the intercept value is about 0.

[0344] In some embodiments, the method has a precision determined by calculating the coefficient of variation (CV), wherein the CV is less than about 10.0%. The CV is less than about 6%. In some embodiments, the CV is less than about 4%. In some embodiments, the CV is less than about 2%. In some embodiments, the CV is less than about 1%.

[0345] In some embodiments, the AR is antibody-mediated rejection (ABMR). In some embodiments, the AR is T-cell-mediated rejection (TCMR).

[0346] Further disclosed herein are methods for detecting transplant donor-derived cell-free DNA (dd-cfDNA) 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 recipient has undergone a transplant selected from a kidney transplant, a liver transplant, a pancreas transplant, a pancreatic 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 xenotransplant recipient has undergone a SPK transplant. In some embodiments, the method can be performed on the xenotransplant recipient on the day of transplant surgery or after transplant surgery, or up to one year after transplant surgery.

[0347] In some embodiments, disclosed herein are methods for amplifying target loci in donor-derived cell-free DNA (dd-cfDNA) from a blood sample of a xenotransplant recipient, the method comprising: a) extracting DNA from the 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 target loci, wherein the target loci comprise between 50 and 5,000 target loci, including polymorphic and non-polymorphic loci; and c) amplifying the target loci.

[0348] In some embodiments, disclosed herein are methods for detecting donor-derived cell-free DNA (dd-cfDNA) in a blood sample from a transplant recipient, the method comprising: (a) extracting DNA from the blood sample from the transplant recipient, the DNA including cell-free DNA derived from both transplanted cells and the transplant recipient; (b) enriching the extracted DNA at target loci, the target loci including 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; (c) amplifying the target loci; (d) contacting the amplified target loci with probes that specifically hybridize to the target loci; and (e) detecting binding of the probes to the target loci, thereby detecting dd-cfDNA in the blood sample. In some embodiments, the probes are labeled with a detectable marker.

[0349] In some embodiments, disclosed herein are methods for detecting the likelihood of xenograft rejection in a transplant recipient, the method comprising: a) extracting DNA from a blood sample of the xenograft recipient, wherein the DNA includes cell-free DNA from both the transplanted cells and the xenograft recipient; b) enriching the extracted DNA at target loci, wherein the target loci include 50 to 5,000 target loci, including polymorphic 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 blood sample, wherein a higher amount of dd-cfDNA indicates a higher likelihood of xenograft rejection.

[0350] In some embodiments, disclosed herein is a method for diagnosing acute rejection of a xenograft in a xenograft recipient, the method comprising: a) extracting DNA from a blood sample of the transplant recipient, wherein the DNA includes cell-free DNA from both the transplanted cells and the xenograft recipient; b) enriching the extracted DNA at target loci, wherein the target loci include 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplant DNA and the amount of recipient DNA in the recipient 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 acute rejection of the xenograft.

[0351] In some embodiments, in the methods disclosed herein, the xenograft rejection is antibody-mediated transplant rejection. In some embodiments, the 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 will undergo borderline rejection, other damage, or is stable.

[0352] In some embodiments, disclosed herein are methods for monitoring immunosuppressive therapy in a subject, the methods comprising: a) extracting DNA from a blood sample of a transplant recipient, the DNA including cell-free DNA from both transplanted cells and the transplant recipient; b) enriching the extracted DNA at target loci, the target loci including 50 to 5,000 target loci, including polymorphic and non-polymorphic loci; c) amplifying the target loci; and d) measuring the amount of transplant DNA and the amount of recipient DNA in the recipient blood sample, wherein a change in the level of dd-cfDNA over a time interval indicates xenograft status. In some embodiments, the method further comprises adjusting the immunosuppressive therapy based on the level of dd-cfDNA over a time interval. In some embodiments, an increase in the level of dd-cfDNA indicates xenograft rejection and the need for adjustment of immunosuppressive therapy. In some embodiments, a change or decrease in the level of dd-cfDNA indicates transplant tolerance or stability and the need for adjustment of immunosuppressive therapy.

[0353] In some embodiments, in the methods disclosed herein, the target locus is amplified with an amplicon about 50-100 bp in length, or about 60-80 bp in length, in some embodiments, the amplicon is about 65 bp in length.

[0354] Analysis method In some embodiments, the method also includes obtaining genotype data from one or both of the xenotransplant donor and the xenotransplant recipient. In some embodiments, obtaining genotype data from one or both of the xenotransplant donor and the xenotransplant recipient includes preparing DNA from the donor and the recipient, the preparing including preferentially enriching 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.

[0355] In some embodiments, constructing a joint distribution model of expected allele count probabilities for multiple polymorphic loci on a chromosome is performed using genotype data obtained from one or both of the transplant donor and the transplant recipient. In some embodiments, the first sample is isolated from the plasma of the transplant recipient, and obtaining the genotype data from the transplant recipient is performed by estimating the recipient's genotype data from DNA measurements performed on the prepared sample.

[0356] In some embodiments, the preferential enrichment results in an average degree of allelic bias between the prepared sample and the first sample of a factor selected from the group consisting of 2-fold or less, 1.5-fold or less, 1.2-fold or less, 1.1-fold or less, 1.05-fold or less, 1.02-fold or less, 1.01-fold or less, 1.005-fold or less, 1.002-fold or less, 1.001-fold or less, and 1.0001-fold or less. In some embodiments, the plurality of polymorphic loci are SNPs. In some embodiments, measuring the DNA or RNA in the prepared sample is performed by sequencing.

[0357] In some embodiments, a diagnostic box is disclosed to aid in determining the xenograft status of a xenograft recipient, where the diagnostic box can perform the preparation and measurement steps of the disclosed methods.

[0358] In some embodiments, the allele counts are probabilistic rather than binary. In some embodiments, measurements of DNA from samples prepared at multiple polymorphic loci are also used to determine whether a xenograft has inherited one or more linked haplotypes.

[0359] In some embodiments, constructing a joint distribution model of allele count probabilities is done by modeling the dependency between polymorphic alleles on a chromosome using data on the probability of chromosome crossover at different locations on the chromosome. In some embodiments, constructing a joint distribution model of allele counts and determining the relative probability of each hypothesis is done using a method that does not require the use of a reference chromosome.

[0360] In some embodiments, determining the relative probability of each hypothesis utilizes the estimated proportion 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 genotype data. In some embodiments, selecting the transplant state corresponding to the hypothesis with the greatest probability is performed using maximum likelihood estimation or maximum a posteriori estimation.

[0361] In some embodiments, calling the xenograft status also includes combining the relative probability and allele count probability of each status hypothesis determined using the joint distribution model with the relative probability of each status hypothesis calculated using statistical methods derived from the group consisting of read count analysis, heterozygosity rate comparisons, statistics available only using donor genetic information, normalized genotype signal probabilities for a particular donor / recipient context, statistics calculated using estimated engraftment fractions for the first sample or prepared samples, and combinations thereof.

[0362] In some embodiments, a reliability estimate is calculated for the called xenograft status. In some embodiments, the method also includes taking a clinical action based on the called xenograft status.

[0363] In some embodiments, a report indicating the determined xenograft status is generated using this method. In some embodiments, a kit for determining xenograft status designed for use with the methods disclosed herein is disclosed, the kit including a plurality of inner forward primers and optionally a plurality of inner reverse primers, each primer designed to hybridize to a DNA region immediately upstream and / or downstream from one of the polymorphic sites on the target chromosome, and optionally to additional chromosomes, wherein the hybridization region is separated from the polymorphic site by a small number of bases, the small number being selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-60, and combinations thereof.

[0364] In some embodiments, the cutoff threshold takes into account one or more of donor genome copies per volume of plasma, cell-free DNA yield per volume of plasma, donor height, donor weight, donor age, donor sex, donor ethnicity, donor organ weight, donor organ, living vs. deceased donor, related vs. unrelated donor, recipient height, recipient weight, recipient age, recipient sex, recipient ethnicity, creatinine, eGFR (estimated glomerular filtration rate), cfDNA methylation, DSA (donor-specific antibodies), KDPI (Kidney Donor Profile Index), medications (immunosuppressants, steroids, anticoagulants, etc.), infections (BKV, EBV, CMV, UTI), recipient and / or donor HLA allele or epitope mismatch, Banff classification of renal allograft pathology, and cause-specific vs. surveillance or protocol biopsy.

[0365] In some embodiments, the cutoff threshold is scaled according to the amount of total cfDNA in the blood sample.

[0366] In some embodiments, the method has a sensitivity of at least 80% with a confidence interval of 95% in identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0367] In some embodiments, the method has a specificity of at least 70% with a confidence interval of 95% in identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0368] In some embodiments, the method has a sensitivity of at least 80% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 85% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 90% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a sensitivity of at least 95% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0369] In some embodiments, the method has a specificity of at least 70% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 75% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 85% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has a specificity of at least 90% and a confidence interval of 95% for identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample. In some embodiments, the method has at least 95% specificity, with a 95% confidence interval, in identifying acute rejection (AR) over non-AR when the amount of dd-cfDNA is above a cutoff threshold scaled according to the amount of total cfDNA in the blood sample.

[0370] Multiplex Amplification In some embodiments, the method includes performing a multiplex amplification reaction to amplify multiple target loci in one reaction mixture prior to sequencing the selectively enriched RNA or DNA.

[0371] In certain exemplary embodiments, the nucleic acid sequence data is generated by performing high-throughput RNA sequencing of multiple copies of a series of amplicons generated using a multiplex amplification reaction, where each amplicon in the series spans at least one polymorphic locus in a set of polymorphic loci, and each polymorphic locus in the set is amplified. In certain exemplary embodiments, the nucleic acid sequence data is generated by performing high-throughput DNA sequencing of multiple copies of a series of amplicons generated using a multiplex amplification reaction, where each amplicon in the series spans at least one polymorphic locus in a set of polymorphic loci, and each polymorphic locus in the set is amplified. For example, in these embodiments, multiplex PCR may be performed that amplifies amplicons across 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). The 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 massively multiplexed PCR disclosed herein, provide exemplary processes for performing amplification reactions to help achieve improved multiplexing and, therefore, sensitivity levels.

[0372] In some embodiments, the amplification is performed using direct multiplex PCR, sequential PCR, nested PCR, double nested PCR, one-and-a-half PCR, or any combination thereof. sided nested PCR, fully nested PCR, one-sided fully nested PCR, one-sided nested PCR, hemi-nested PCR, hemi-nested PCR, triplex hemi-nested PCR, semi-nested PCR, one-sided semi-nested PCR, reverse semi-nested PCR, or one-sided PCR, as described in U.S. Application No. 13 / 683,604, filed November 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed November 18, 2011, U.S. Publication No. 2012 / 0270212, and U.S. Serial No. 61 / 994,791, filed May 16, 2014, which are incorporated by reference in their entireties.

[0373] In some embodiments, multiplex PCR is used. In some embodiments, a method for amplifying target loci in a nucleic acid sample includes (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, or 100,000 different target loci to generate a single reaction mixture, and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to generate amplified products containing target amplicons. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplification products are primer dimers. In some embodiments, the primers are in solution (e.g., dissolved in a liquid phase rather than a solid phase). In some embodiments, the primers are in solution and not immobilized on a solid support. In some embodiments, the primers are not part of a microarray.

[0374] In certain embodiments, the multiplex amplification reaction is performed under limiting primer conditions for at least half of the reactions. In some embodiments, limiting primer concentrations are used in 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or all of the reactions in the multiplex reaction. Factors to consider in achieving limiting primer conditions in amplification reactions such as PCR are provided herein.

[0375] For certain embodiments, multiplex amplification reactions may include, for example, 2,500 to 50,000 multiplex reactions. In certain embodiments, multiplex reactions ranging from 100, 200, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25,000, and 50,000 at the lower end of the range to 200, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25,000, 50,000, and 100,000 at the higher end of the range are performed.

[0376] In one embodiment, multiplex PCR assays are designed to amplify potentially heterozygous SNP loci or other polymorphic or non-polymorphic loci on one or more chromosomes, and these assays are used in a single reaction to amplify DNA. The number of PCR assays can be 50-200 PCR assays, 200-1,000 PCR assays, 1,000-5,000 PCR assays, or 5,000-20,000 PCR assays (50-200 reactions, 200-1,000 reactions, 1,000-5,000 reactions, 5,000-20,000 reactions, and more than 20,000 reactions, respectively). In one embodiment, a multiplex pool of at least about 10,000 PCR assays (10,000 reactions) is designed to amplify potentially heterozygous SNP loci in a single reaction and amplify cfDNA obtained from blood, plasma, serum, solid tissue, or urine samples. The SNP frequency of each locus can be determined by clonal methods or some other method of sequencing the amplicon. In another embodiment, the original cfDNA sample is split into two samples and 5,000 parallel assays are performed. In another embodiment, the original cfDNA sample is split into n samples and approximately 10,000 / n parallel assays are performed, where n is 2 to 12, or 12 to 24, or 24 to 48, or 48 to 96.

[0377] In one embodiment, the method disclosed herein uses highly efficient, highly multiplexed targeted PCR to amplify DNA, followed by high-throughput sequencing to determine the allele frequency at each target locus. One technique that allows highly multiplexed targeted PCR to be performed in a highly efficient manner involves designing primers that are unlikely to hybridize with each other. PCR probes, typically called primers, are selected by creating a thermodynamic model of potentially adverse interactions between at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 potential primer pairs or unintended interactions between primers and sample DNA, and then using this model to eliminate designs that are incompatible with other designs in the pool. Another technique that allows highly multiplexed targeted PCR to be performed in a highly efficient manner is to use a partial or full nesting approach to targeted PCR. Using one or a combination of these approaches allows for the multiplexing of at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 primers in a single pool, with the resulting amplified DNA containing the majority of DNA molecules, when sequenced, mapping to the target locus. Using one or a combination of these techniques allows for the multiplexing of large numbers of primers in a single pool, with the resulting amplified DNA containing more than 50%, more than 80%, more than 90%, more than 95%, more than 98%, or more than 99% of the DNA molecules mapping to the target locus.

[0378] Bioinformatics methods are used to analyze the genotype data obtained from the multiplex PCR. Bioinformatics methods useful and relevant to the methods disclosed herein can be found in U.S. Patent Publication No. 2018 / 0025109, which is incorporated herein by reference.

[0379] High-throughput sequencing In some embodiments, the sequence of the amplicon is determined by performing high-throughput sequencing.

[0380] Genotype data of transplant recipients and / or transplant donors can be converted from molecular to electronic states by measuring the appropriate genetic material using tools and techniques from a group including, but not limited to, genotyping microarrays and high-throughput sequencing. Some high-throughput sequencing methods include Sanger DNA sequencing, pyrosequencing, the ILLUMINA SOLEXA platform, ILLUMINA's GENOME ANALYZER, or APPLIED BIOSYSTEM's 454 sequencing platform, HELICOS' TRUE SINGLE MOLECULE SEQUENCING platform, HALCYON MOLECULAR's electron microscope sequencing, PacBio, Oxford Nanopore, or any other sequencing method. In some embodiments, high-throughput sequencing is performed on an Illumina NEXTSEQ®. All of these methods physically convert the genetic data stored in a sample of DNA into a set of genetic data that is typically stored in a memory device during processing.

[0381] In some embodiments, the sequence of the selectively enriched DNA is determined by performing a microarray analysis. In one embodiment, the microarray may be an ILLUMINA SNP microarray or an AFFYMETRIX SNP microarray.

[0382] In some embodiments, the sequence of the selectively enriched DNA is determined by quantitative PCR (qPCR) or digital droplet PCR (ddPCR) analysis. qPCR measures the intensity of fluorescence at a specific time (generally per amplification cycle) to determine the relative amount of target molecules (DNA). ddPCR measures the actual number of molecules (target DNA) because each molecule is in a droplet, thus providing a separate "digital" measurement. This provides absolute quantification because ddPCR measures the sample's positive fraction, i.e., the number of droplets that fluoresce due to proper amplification. This positive fraction accurately indicates the initial amount of template nucleic acid.

[0383] Tracer DNA and its uses Tracer DNA for estimating the amount of total cfDNA in a sample is described in U.S. Provisional Application No. 63 / 031,879, filed May 29, 2020, entitled "Improved Methods for Detection of Donor-Derived Cell-Free DNA," the entire contents of which are incorporated herein by reference. 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.

[0384] In some embodiments, the tracer DNA comprises a DNA molecule having a length of about 50 to 500 bp, or about 75 to 300 bp, or about 100 to 250 bp, or about 125 to 200 bp, or about 125 bp, or about 160 bp, or about 200 bp, or about 500 to 1,000 bp.

[0385] In some embodiments, the tracer DNA comprises DNA molecules having the same or substantially the same length, for example, DNA molecules having a length of about 125 bp, about 160 bp, or about 200 bp. In some embodiments, the tracer DNA comprises DNA molecules having different lengths, for example, 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 cell-free DNA in a sample.

[0386] In some embodiments, the tracer DNA comprises a target sequence, and the target sequence comprises a barcode located between a pair of primer binding sites that can bind to a pair of primers. 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. During the mmPCR target enrichment step, the primer pair targeting the SNP locus can also amplify a portion of the tracer DNA that includes the barcode.

[0387] In some embodiments, the barcode is any barcode. In some embodiments, the barcode comprises the reverse complement of a corresponding endogenous genomic sequence that is amplifiable by the same primer pair.

[0388] In some embodiments, the target sequence in the tracer DNA is flanked on one or both sides by endogenous genomic sequences. In some embodiments, the target sequence in the tracer DNA is flanked on one or both sides by non-endogenous sequences.

[0389] In some embodiments, the tracer DNA comprises multiple target sequences. In some embodiments, the tracer DNA comprises a first target sequence comprising a first barcode located between a first pair of primer binding sites capable of binding to a first pair of primers and a second barcode located 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 loci with the barcode. In some embodiments, the first and / or second barcodes are any barcode. In some embodiments, the first and / or second barcodes comprise the reverse complement of a corresponding endogenous genomic sequence that is amplifiable by the first or second primer pair. In some embodiments, the first and / or second target sequences in the tracer DNA are flanked on one or both sides by endogenous genomic sequences. In some embodiments, the first and / or second target sequence in the tracer DNA is flanked on one or both sides by non-endogenous sequences.

[0390] In some embodiments, the tracer DNA comprises DNA molecules with the same or substantially the same sequence. In some embodiments, the tracer DNA comprises DNA molecules with different sequences.

[0391] In some embodiments, the tracer DNA comprises a first DNA comprising a first target sequence and a second DNA comprising a second target sequence. In some embodiments, the first target sequence and the second target sequence have different barcodes located between the same primer binding sites. In some embodiments, the first target sequence and the second target sequence have different barcodes located between the same primer binding sites, and 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 located between the same primer binding sites, and 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 therefore contain different primer binding sites. In some embodiments, the amount of the first DNA and the amount of the second DNA are the same or substantially the same in the tracer DNA. In some embodiments, the amount of the first DNA and the amount of the second DNA are different in the tracer DNA.

[0392] In certain embodiments, tracer DNA can be used to improve the accuracy and precision of the methods described herein, aid in quantification over a wider input range, assess the efficiency of different steps across different size ranges, and / or calculate the fragment size distribution of the input material.

[0393] 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 the biological sample, wherein a first tracer DNA is added before or after 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 obtained from the first tracer DNA.

[0394] In some embodiments, the method comprises adding a first tracer DNA to a whole blood sample prior to plasma extraction. In some embodiments, the method comprises adding a first tracer DNA to a plasma sample after plasma extraction and prior to isolation of cell-free DNA. In some embodiments, the method comprises adding the first tracer DNA to a composition comprising isolated cell-free DNA. In some embodiments, the method comprises ligating an adapter to the isolated cell-free DNA to obtain a composition comprising adapter-ligated DNA, and adding the first tracer DNA to the composition comprising adapter-ligated DNA.

[0395] In some embodiments, the method further comprises adding a second tracer DNA prior to target amplification, hi some embodiments, the method further comprises adding a second tracer DNA after target amplification.

[0396] In some embodiments, the amount of total cfDNA in a sample is estimated using the NOR of the tracer DNA (identifiable by barcode), 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 can also be used in conjunction with the plasma volume 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 donor cfDNA and donor cfDNA per plasma volume.

[0397] Thus, 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 isolation of the cell-free DNA; b) performing target 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 obtained from the first tracer DNA composition.

[0398] 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 isolation of the cell-free DNA; b) performing target 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 obtained from the first tracer DNA composition.

[0399] In a further aspect, the present invention relates to a method for determining the occurrence or likelihood of occurrence of transplant rejection, the method comprising: a) isolating cell-free DNA from a biological sample of a 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 isolation of the cell-free DNA; b) performing target 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 obtained from the first tracer DNA composition; and determining the occurrence or likelihood of occurrence of transplant rejection using the amount of donor-derived cell-free DNA by comparing the amount of donor-derived cell-free DNA with a threshold value, wherein the threshold value is determined according to the amount of total cell-free DNA.

[0400] In some embodiments, the threshold is a function of the number of sequencing reads of cell-free DNA from the donor.

[0401] In some embodiments, the method further comprises flagging the sample if the amount of total cell-free DNA is outside a predetermined range. 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.

[0402] In some embodiments, the method comprises adding a first tracer DNA composition to a whole blood sample prior to plasma extraction. In some embodiments, the method comprises adding a first tracer DNA composition to a plasma sample after plasma extraction and prior to isolation of cell-free DNA. In some embodiments, the method comprises adding the first tracer DNA composition to a composition comprising isolated cell-free DNA. In some embodiments, the method comprises ligating an adapter to the isolated cell-free DNA to obtain a composition comprising adapter-ligated DNA, and adding the first tracer DNA composition to the composition comprising adapter-ligated DNA.

[0403] In some embodiments, the method further comprises adding a second tracer DNA composition prior to target amplification, hi some embodiments, the method further comprises adding a second tracer DNA composition after target amplification.

[0404] In some embodiments, the first and / or second tracer DNA compositions comprise a plurality of DNA molecules having different sequences.

[0405] In some embodiments, the first and / or second tracer DNA compositions comprise a plurality of DNA molecules having different concentrations.

[0406] In some embodiments, the first and / or second tracer DNA compositions comprise a plurality of DNA molecules having different lengths. In some embodiments, a plurality of DNA molecules having different lengths is used to determine the size distribution of cell-free DNA in a sample.

[0407] In some embodiments, the first and / or second tracer DNA compositions comprise a plurality of DNA molecules of non-human origin.

[0408] In some embodiments, the first and / or second tracer DNA compositions each comprise a target sequence, the target sequence comprising a barcode located between a pair of primer binding sites capable of binding to one of the primer pairs, hi some embodiments, the barcode comprises the reverse complement of a corresponding endogenous genomic sequence that is amplifiable by the same primer pair.

[0409] In some embodiments, the ratio between the number of tracer DNA reads and the number of sample DNA reads is used to quantify the amount of total cell-free DNA. In some embodiments, the ratio between the number of barcode reads and the number of corresponding endogenous genomic sequence reads is used to quantify the amount of total cell-free DNA.

[0410] In some embodiments, the target sequence is flanked on one or both sides by endogenous genomic sequence. In some embodiments, the target sequence is flanked on one or both sides by non-endogenous sequence.

[0411] In some embodiments, the first and / or second tracer DNA compositions comprise synthetic double-stranded DNA molecules. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of 50-500 bp. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of 75-300 bp. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of 100-250 bp. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of 125-200 bp. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of approximately 200 bp. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of approximately 160 bp. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of approximately 125 bp. In some embodiments, the first and / or second tracer DNA compositions comprise DNA molecules having a length of 500 to 1,000 bp.

[0412] In some embodiments, the target amplification comprises amplifying at least 100 polymorphism or SNP loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 200 polymorphism or SNP loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 500 polymorphism or SNP loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 1,000 polymorphisms or SNP loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 2,000 polymorphisms or SNP loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 5,000 polymorphisms or SNP loci in a single reaction volume. In some embodiments, the target amplification comprises amplifying at least 10,000 polymorphisms or SNP loci in a single reaction volume.

[0413] In some embodiments, each primer pair is designed to amplify a target sequence of about 35 to 200 bp. In some embodiments, each primer pair is designed to amplify a target sequence of about 50 to 100 bp. In some embodiments, each primer pair is designed to amplify a target sequence of about 60 to 75 bp. In some embodiments, each primer pair is designed to amplify a target sequence of about 65 bp.

[0414] 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.

[0415] In some embodiments, the transplant is a human transplant. In some embodiments, the transplant is a porcine transplant. In some embodiments, the transplant is a transplant from a non-human animal.

[0416] In some embodiments, the transplant is an organ transplant, a tissue transplant, or a cell transplant, hi some embodiments, the transplant is a kidney transplant, a liver transplant, a pancreas transplant, an intestinal transplant, a heart transplant, a lung transplant, a heart / lung transplant, a stomach transplant, a testis transplant, a penis transplant, an ovary transplant, a uterus transplant, a thymus transplant, a face transplant, a hand transplant, a leg transplant, a bone transplant, a bone marrow transplant, a cornea transplant, a skin transplant, a pancreatic islet cell transplant, a heart valve transplant, a blood vessel transplant, or a blood transfusion.

[0417] In some embodiments, the method further comprises determining the transplant rejection as antibody-mediated transplant rejection, T-cell-mediated transplant rejection, graft injury, viral infection, bacterial infection, or borderline rejection. [Example]

[0418] Example 1 This example is illustrative only, and those skilled in the art will appreciate that the invention disclosed herein can be practiced in a variety of other ways.

[0419] blood sample Female adult or young adult patients receive donor organs from related or unrelated living donors or unrelated deceased donors. Blood sampling for patients after transplant surgery occurs at the time of allograft biopsy or at various pre-specified time intervals based on laboratory protocols. Typically, samples are matched for biopsy, and blood is collected at the time of clinical dysfunction and biopsy or at the time of protocol biopsy (at which point most patients were clinically dysfunctional). Additionally, some patients undergo serial blood sampling after transplantation. Test sample selection is based on (a) the availability of adequate plasma and (b) whether the sample is linked to biopsy information. Of the complete 300-sample cohort, 72.3% were collected on the day of biopsy.

[0420] Measurement of nucleic acids in blood samples Nucleic acids such as RNA or DNA, particularly cell-free DNA, mRNA, and microRNA, were extracted from plasma samples using the QIAAMP™ Circulating Nucleic Acid Kit (Qiagen), and the LABCHIP™ NGS 5k kit (Perkin Elmer, Waltham, Massachusetts, USA) was used for quantification. Library preparation was performed using the Natera Library Prep kit, as described in Abbosh et al., Nature 545:446-451 (2017), with 18 cycles of library amplification modified to plateau the library. The purified library was quantified using the LABCHIP™ NGS 5k, as described in Abbosh et al., Nature 545:446-451 (2017). Target enrichment was achieved using massively multiplexed PCR (mmPCR) targeting 13,392 single nucleotide polymorphisms (SNPs) using a modified version of the method described in Zimmermann et al., Prenat. Diagn. 32:1233-1241 (2012). Amplicons were then single-end sequenced for 50 cycles on an Illumina HiSeq 2500 RAPIDRUN® with 10-11 million reads per sample.

[0421] Statistical analysis of nucleic acids, dd-cfDNA, and eGFR In each sample, donor-derived RNA and / or dd-cfDNA are measured and correlated with rejection status, and the results are compared to eGFR. All statistical tests are two-sided, where applicable. Significance is set at p<0.05. Because the distribution of dd-cfDNA in patients is severely skewed between groups, data are analyzed using the Kruskal-Wallis rank sum test followed by Dunn's multiple comparison test with Holm correction. eGFR (serum creatinine in mg / dL) is calculated as previously described for adult and pediatric patients. Briefly, eGFR = 186 × serum creatinine -1.154 ×Age -0.203 × (1.210 for blacks) × (0.742 for women).

[0422] Contaminations can be removed using Cas9 / Cas12a or Cas13 CRISPR technology. 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.

[0423] Example 2 In this example, the xenotransplant monitoring tests disclosed herein were evaluated. Figure 1 shows an overview of one of the test methods for detecting cell-free DNA from animal donors in human plasma samples.

[0424] First, cell-free DNA (cfDNA) was purified from human and pig plasma samples. Next, combinations of pig and human cell-free DNA were created. Combinations were tested, ranging from 100% to 0.01% pig cell-free DNA. Sequencing libraries were prepared from the combined pig and human cfDNA.

[0425] The porcine / human cfDNA ratio for each combination was determined using whole-genome sequencing of the prepared libraries, and the determined porcine / human cfDNA ratios were plotted against the actual ratio for each sample, as shown in Figure 2. Linear regression analysis revealed a linear relationship (R ) between the measured and actual porcine / human cfDNA ratios. 2 = 0.9961). Therefore, this evaluation provides proof of concept for measuring the amount of cfDNA from xenografts using whole-genome sequencing of cfDNA extracted from plasma samples, which is useful for monitoring xenograft rejection. * * * *

Claims

1. 1. A method for preparing a non-naturally occurring composition of DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, 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 cell-free DNA from the animal donor 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 cutoff threshold indicative of xenograft rejection or damage.

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

3. 1. A method for preparing a non-naturally occurring composition of amplified DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, 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 a composition of amplified DNA by performing targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume, wherein the target loci include both human and animal target loci; sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of both donor-derived cell-free DNA and xenotransplant recipient-derived cell-free DNA based on the sequencing reads, wherein the human and animal loci are the same and the human and animal reads are distinguished based on insertion sequences; (c) determining whether the proportion of cell-free DNA from the animal donor, or a function thereof, exceeds a cutoff threshold indicative of xenograft rejection.

4. 1. A method for preparing a non-naturally occurring composition of amplified DNA from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, 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 composition of amplified DNA by performing targeted amplification of the extracted cell-free DNA at 10 to 50,000 target loci in a single reaction volume, wherein the target loci include one or more target loci that exhibit xenograft rejection; (c) 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 cutoff threshold indicative of xenograft rejection.

5. The method of claims 1 to 4, wherein the composition of DNA comprises one or more target loci indicative of xenograft rejection, and the determining step further comprises determining an amount of the one or more target loci indicative of xenograft rejection and determining whether the amount or function thereof of the one or more target loci indicative of xenograft rejection exceeds a cutoff threshold indicative of xenograft rejection, wherein xenograft rejection is determined by a combination of (i) the amount or function thereof of the one or more target loci indicative of xenograft rejection and (ii) the total amount of cell-free DNA from the animal donor or the percentage of cell-free DNA from the animal donor.

6. 1. A method for preparing a non-naturally occurring composition of complementary DNA (cDNA) from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, comprising: (a) extracting RNA from a 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 RNA from the donor based on the sequencing reads; (c) determining whether the total amount of donor-derived RNA from the xenograft, or a function thereof, exceeds a cutoff threshold indicative of xenograft rejection.

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

8. 1. A method for preparing a non-naturally occurring composition of complementary DNA (cDNA) amplified from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, 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 composition of amplified cDNA from the extracted RNA by performing targeted amplification of cDNA at 10 to 50,000 target loci in a single reaction volume, wherein the target loci include both human loci and target loci; sequencing the amplified cDNA by high-throughput sequencing to obtain sequencing reads; and quantifying the amount of both donor-derived RNA and xenotransplant recipient-derived RNA based on the sequencing reads, wherein the human loci and animal loci are the same and the human reads and animal reads are distinguished based on insert sequences; (c) determining whether the proportion of donor-derived RNA, or a function thereof, exceeds a cutoff threshold indicative of xenograft rejection.

9. 1. A method for preparing a non-naturally occurring composition of complementary DNA (cDNA) amplified from RNA extracted from a blood, plasma, serum, or urine sample of a xenotransplant recipient, useful for determining xenotransplant rejection, comprising: (a) extracting RNA from the blood, plasma, serum, or urine sample of the xenotransplant recipient, wherein the extracted RNA comprises donor-derived cell-free RNA and recipient-derived RNA; (b) preparing a composition of amplified cDNA derived from the extracted RNA by performing targeted amplification of cDNA at 10 to 50,000 target loci in a single reaction volume, the target loci including one or more target loci exhibiting xenograft rejection; (c) 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 cutoff threshold indicative of xenograft rejection.

10. The method of claims 6 to 9, wherein the composition comprises target loci indicative of xenograft rejection, and the determining step further comprises determining an amount of the one or more target loci indicative of xenograft rejection and determining whether the amount or function thereof of the one or more target loci indicative of xenograft rejection exceeds a cutoff threshold indicative of xenograft rejection, and xenograft rejection is determined by a combination of the amount or function thereof of the one or more target loci indicative of xenograft rejection and the total amount of RNA derived from the animal donor or the proportion of RNA derived from the animal donor.

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

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

13. 1. A method of administering immunosuppressive therapy in a xenotransplant recipient, comprising: (a) measuring the amount of donor-derived protein according to claim 11; (b) titrating the dosage of said immunosuppressive therapy according to said amount of donor-derived protein or a function thereof.

14. 1. A method of administering immunosuppressive therapy in a xenotransplant recipient, comprising: (a) measuring the amount of one or more target proteins according to claim 12; (b) titrating the dosage of said immunosuppressive therapy according to said amount or function thereof of said one or more target proteins.

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

16. 16. The method of claim 15, further comprising titrating the dosage of immunosuppressive therapy in response to the longitudinal changes in the donor-derived protein, donor-derived target protein, or function thereof.

17. 17. The method of 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, comprising extracting proteins from extracellular vesicles (EVs) isolated from the blood, plasma, serum, or urine sample of the xenotransplant recipient.

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

19. The amount of cell-free DNA from the donor is 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 to 50,000 target loci in a single reaction volume; 20. The method of claim 18, wherein the amount of cell-free DNA from the donor is measured by sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of cell-free DNA from the donor based on the sequencing reads.

20. 20. The method of claim 18 or 19, further comprising repeating steps (a)-(b) longitudinally on 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. 21. The method of claim 20, further comprising titrating the dosage of immunosuppressive therapy in response to the longitudinal change in the total amount of, or a function thereof, of cell-free DNA and the longitudinal change in the amount of, or a function thereof, of donor-derived cell-free DNA.

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

23. The amount of RNA from the donor is 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 a composition of amplified complementary DNA (cDNA) derived from said extracted RNA by performing multiplexed target amplification of cDNA at 200-50,000 animal target loci in a single reaction volume, and detecting and quantifying said amount of RNA from animal donors; 21. The method of claim 20, wherein the amount of RNA from the donor is measured by sequencing the amplified cDNA by high-throughput sequencing to obtain sequencing reads, and quantifying the amount of RNA from the donor based on the sequencing reads.

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

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

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

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

28. 26. The method of 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. 29. The method of claim 28, wherein the sequencing comprises next-generation whole genome sequencing.

30. The method of 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. 26. The method of 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 ratiometric and / or machine learning-artificial intelligence comparisons at single or multiple time points.

32. 26. The method of 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 microscopic imaging.

33. 33. The method of any one of claims 1 to 10 and 18 to 32, wherein the target locus comprises a single nucleotide polymorphism (SNP).

34. The method of any one of claims 1 to 10 and 18 to 33, wherein the cutoff threshold is an estimated ratio of donor-derived cell-free DNA or RNA to 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 cutoff threshold is proportional to the absolute value of the cell-free DNA concentration or RNA concentration derived from the donor.

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

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

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

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

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

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

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

43. 43. The method of any one of claims 6-10 and 22-42, further comprising utilizing CRISPR-Cas to target and deplete contaminating or excess nucleic acid species in the sample, thereby increasing the proportion of desired reads that map to target loci of interest per sample and sample throughput per sequencing run.

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

45. 44. The method of claim 43, wherein the contaminating or excess nucleic acid species comprise hemoglobin mRNA, tRNA, rRNA, miR-451, miR-144, and miR-486.

46. 46. ​​The method of claims 43-45, further comprising depleting adapter dimers, primer dimers, and unwanted ligation products from a composition of amplified nucleic acid comprising target loci, thereby increasing the proportion of desired reads that map to target loci of interest per sample and sample throughput per sequencing run.

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

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

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

50. 50. The method of any one of claims 1 to 49, wherein the xenograft recipient is a human subject.

51. 51. The method of any one of claims 1 to 50, wherein the xenograft recipient is receiving one or more xenograft organs selected from pancreas, kidney, liver, heart, lung, intestine, thymus, and uterus.

52. 52. The method of any one of claims 1 to 51, wherein the sample is obtained from the xenotransplant recipient within 18 months after transplantation.

53. 53. The method of any one of claims 1 to 52, wherein the xenotransplant recipient's risk of rejection is determined using logistic regression, random forest, or decision tree machine learning analysis.

54. 54. The method of claim 53, wherein the logistic regression, random forest, or decision tree machine learning analysis further incorporates one or more parameters selected from time since transplant, age of the xenograft recipient and / or xenograft donor, and gender of the xenograft recipient and / or xenograft donor.

55. 55. The method of any one of claims 1 to 54, wherein the xenograft is derived from a pig, a primate, a baboon, a cow, or a dog.

56. 56. The method of any one of claims 1 to 55, wherein the xenograft is of porcine origin.

57. 57. The method of 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 a xenotransplant recipient.

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