Nucleic acid analysis of perfusion or irrigation fluids

Abstract

Provided herein are systems and methods for preparing formulations of non-naturally occurring DNA from perfusate or rinse fluids that can be used to assess transplantation outcomes of donor organs or donor tissues perfused with the perfusate or prepared with rinse fluids for transplantation. The formulation can also be used to assess the quality of the organ or tissue. The analysis of the DNA can be used to make clinical decisions. The analysis of the DNA may also be used to adaptively control a mechanical perfusion system for preserving the organ or tissue by generating and performing appropriate parameter adjustments based on the characteristics of the sampled DNA.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 413,834, filed on October 6, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] In the field of organ transplantation, it is generally necessary to preserve organs in a controlled manner after removal from a donor and before transplantation into a recipient in order to maintain organ viability and prevent organ damage and transplant failure. Common preservation methods include standard of care static cold storage (SCS) and various dynamic perfusion techniques. Dynamic perfusion of organs attempts to mimic the physiological environment of the human body by circulating a fluid through the organ in a cyclic manner.

[0004] Due to improved outcomes, machine perfusion has increasingly been used in place of SCS, especially for high-risk or marginal organs such as recovered deceased donor kidneys. SCS involves keeping the organ under hypoxic conditions, which leads to metabolite accumulation via anaerobic metabolism. This accumulation of metabolites can increase the risk of ischemia-reperfusion injury (IRI) after reperfusion of the organ in the recipient. Therefore, it is desirable to use a preservation method that can regulate metabolite levels in the organ and re-supply oxygenated blood / oxygen carriers, drugs, and essential nutrients. In addition, machine perfusion at normal temperature allows for pre-transplant reconditioning of the donor organ, such that blood flow can be restored earlier and recipient outcomes improved. However, the perfused organ may still be damaged, resulting in negative transplant outcomes. Thus, there is a need for a method to assess the quality or status of an organ during perfusion prior to transplantation.

[0005] In addition, all solid organs preserved using SCS are flushed with a crystalloid solution in the operating room prior to implantation. The purpose of this flush is to remove blood clots and other organic debris from the donor organ's circulatory system prior to anastomosis with the recipient's circulatory system. Summary of the Invention

[0006] The systems, devices, and methods described herein allow for the analysis of nucleic acids sampled from perfusate (the fluid used for the mechanical perfusion of an organ or tissue) or from organ flush fluid (received from a donor). While both fluids are used for the preservation and / or reconditioning of an organ / tissue during storage or transport prior to transplantation into a recipient, the perfusate can be sampled according to the methods described herein in order to isolate and analyze DNA. The perfusion fluid and the flush fluid are typically discarded, and thus sampling, manipulating, and analyzing these fluids are neither routine nor conventional. Nucleic acids from the perfusate, such as cell-free DNA (cfDNA), cellular DNA, or RNA, can be quantified, enriched to produce a non-native preparation for analysis purposes, sequenced, amplified to produce a non-native preparation for analysis purposes, or otherwise manipulated to produce a non-native preparation for analysis purposes in order to inform about the current state of the organ / tissue being perfused by a physician. Analytical results derived from such preparations, such as cfDNA concentration or fragment size distribution (or a subset thereof), can indicate that the organ / tissue has been injured and may no longer be suitable for transplantation or may require adjustment of the perfusion. Analytical results generated by such preparations can also indicate the quality of the donor organ / tissue, which can be used to assess its suitability for transplantation and to inform the process of allocating the organ / tissue to a suitable recipient. The analytical results can predict primary graft dysfunction after organ / tissue transplantation, predict delayed graft function in kidney transplant recipients, and / or predict overall post-transplant organ function.

[0007] In a first aspect, the present disclosure provides a method for preparing a nucleic acid preparation from perfusate that can be used to predict the transplantation outcome of a donor organ or donor tissue. The method includes obtaining a sample of perfusate from a donor organ or donor tissue that has been perfused with perfusate, the perfusate including nucleic acids; isolating the nucleic acids from the sample; and analyzing the isolated DNA to assess at least one of the amount of nucleic acids in the perfusate, the molecular weight of the DNA in the perfusate, or the fragment size distribution of the nucleic acids in the perfusate.

[0008] In some embodiments, the amount of nucleic acids is the total amount of nucleic acids in the perfusate. In some embodiments, the amount of nucleic acids is the amount of high molecular weight DNA (e.g., greater than 200 base pairs) in the perfusate. In some embodiments, the amount of nucleic acids is the amount of low molecular weight DNA (e.g., less than 200 base pairs) in the perfusate. In some embodiments, the molecular weight of the nucleic acids is the average molecular weight of the nucleic acids in the perfusate.

[0009] In some embodiments, the isolated nucleic acid is cell-free DNA. The method may further include assessing that the cell-free DNA is derived from apoptosis if the cell-free DNA has a molecular weight consistent with nucleosomal DNA. In some embodiments, the isolated nucleic acid is cellular DNA. In some embodiments, the isolated nucleic acid is RNA. The RNA can be RNA present in extracellular vesicles such as exosomes and microvesicles. Vesicles captured from the perfusate can be lysed to release the RNA, which can then be purified in solution using techniques such as filtration, hybrid capture, size selection, or other suitable techniques. The method may further include the step of assessing the amount of the isolated RNA as a biomarker of organ / tissue stability or quality. The method may further include assessing the gene expression profile corresponding to the isolated RNA to evaluate the cellular origin of the RNA. For example, when the gene expression profile is related to immune function, the cellular origin of the RNA is assessed as immune cells. The RNA can be used as a biomarker, but when packaged in vesicles, for example, it can also provide a better source picture (e.g., better characterize cell viability or organ status) compared to free RNA. Due to the role of RNA in gene expression, the analysis of RNA can provide more functional information about the organ or tissue status compared to the analysis of DNA. The isolated RNA can be measured to assess at least one of the type of the RNA (e.g., coding or non-coding) or the sequence of the RNA (e.g., mRNA sequence). The type and sequence can provide information indicating the origin of the RNA found in the perfusate and help inform about the status of the organ or tissue. Non-coding RNA can act as a regulator of expression, so the presence or absence of non-coding RNA can be a predictor of transplant outcome and can be considered complementary or alternative to DNA or coding RNA. Manipulation and analysis of RNA may be more difficult than DNA due to the instability of RNA (due to the single-stranded form of uracil and degradation) and the lower natural RNA concentration. Therefore, additional techniques may be required to accurately analyze RNA from perfusate or wash fluid samples. For example, more selective isolation and extraction methods may be required to account for the lower RNA concentration. To address the instability issue, the RNA can be reverse transcribed to produce complementary DNA (cDNA).

[0010] The method may further include normalizing the amount of nucleic acid relative to at least one of the size, weight, volume, or surface area of the donor organ or donor tissue; perfusion time; perfusion volume; or temperature of the perfusion fluid. The method may include assessing the fragmentation pattern of the nucleic acid. For example, the method may include classifying the isolated nucleic acid into randomly degraded nucleic acid from ruptured cells in the perfusion fluid and nucleic acid from apoptosis of cells in the donor organ or donor tissue. The method may further include obtaining one or more additional samples of the perfusion fluid collected from a different time point than the sample; quantifying the amount of nucleic acid (e.g., cellular DNA, cfDNA, or RNA) in the perfusion fluid of the one or more additional samples; and tracking the amount of nucleic acid in the perfusion fluid over time based on the sample and the one or more additional samples. The nucleic acid may include cellular DNA, cfDNA, RNA, or a combination thereof.

[0011] In a second aspect, provided herein is a method for predicting the outcome of transplantation, the method including preparing a nucleic acid preparation according to the method of the first aspect above and further including hypothesizing a predicted outcome of transplantation of the donor organ or donor tissue based on the amount of nucleic acid.

[0012] In some embodiments, the nucleic acid is cellular DNA from donor-derived cells, and the predicted outcome is based on a predicted favorable or unfavorable prognosis of the cellular DNA, such as the cellular DNA indicating an immune response. In some embodiments, the nucleic acid is cell-free DNA, and the predicted outcome is based on a predicted favorable or unfavorable prognosis of the cell-free DNA, such as the cell-free DNA indicating damage to the donor organ or donor tissue. In some embodiments, the predicted outcome is a predicted unfavorable prognosis, which is for a decision maker not to use the organ for transplantation. In some embodiments, the predicted outcome is at least partially based on a risk determination hypothesis generated via an algorithm that uses the amount of nucleic acid as an algorithm input. In some embodiments, the predicted outcome is hypothesized by a "quality" assessment of the donor organ or donor tissue based on the amount of nucleic acid. Such an assessment may indicate at least one of the following characteristics associated with the transplantation outcome: the presence of delayed graft function, the duration of delayed graft function, the primary non-function rate, and organ function at various time points after transplantation.

[0013] In a third aspect, provided herein is a method for predicting the outcome of transplantation, the method including preparing a nucleic acid preparation according to the method of the first aspect above and further including hypothesizing a predicted outcome of transplantation of the donor organ or donor tissue based on the fragment size distribution (or a subset thereof) of the nucleic acid. In some embodiments, the predicted outcome is at least partially based on a risk determination hypothesis evaluated via an algorithm that uses the fragment size distribution (or a subset thereof) as an algorithm input.

[0014] In some embodiments of the second or third aspect, the predicted result is rejection or non-rejection of the transplant. The predicted result may include one or more of the type of transplant rejection and the time of transplant rejection. The method of the second or third aspect may further include recommending to stop the perfusion of the organ or tissue based on the predicted result. If the amount of nucleic acid analyzed in one or more formulations exceeds the amount of threshold nucleic acid (e.g., a threshold calculated based on the organ / tissue size), perfusion may be recommended to be stopped.

[0015] In some embodiments of any one of the first, second, or third aspect, the method further includes performing targeted genetic analysis on the isolated nucleic acid in one or more formulations to identify genetic features in the nucleic acid. The targeted genetic analysis may include targeted amplification and high-throughput sequencing of at least 50 target loci in the nucleic acid. The targeted genetic analysis can be used to evaluate changes in the cellular processes of homeostasis or of the organ / tissue.

[0016] In a fourth aspect, provided herein is a method for feedback-controlled mechanical perfusion of a donor organ or donor tissue. The method includes perfusing the donor organ or donor tissue in a perfusion chamber containing the donor organ or donor tissue at a first value of perfusion parameters; performing the method of any one of the first, second, or third aspect on the donor organ or donor tissue; appropriately adjusting the perfusion parameters at least in part based on the amount of nucleic acid in the perfusion fluid; and adjusting the perfusion parameters to a second value based on the generated appropriate adjustment.

[0017] In some embodiments, the adjustment is an increase or decrease in the perfusion flow rate. In some embodiments, the perfusion flow rate is decreased to a minimum threshold perfusion rate required to maintain the transplant in a state with a threshold likelihood of being assumed or expected to have transplant rejection. The threshold likelihood may be the maximum likelihood of transplant rejection. In some embodiments, the perfusion parameter is the concentration of at least one component of the perfusion fluid. For example, the at least one component is selected from the group consisting of oxygen, stem cells, immunosuppressive drugs, nutrients, or red blood cells. In some embodiments, the perfusion parameter is the temperature or pH of the perfusion fluid.

[0018] The method may further include collecting a sample of the adjusted perfusion fluid; and evaluating whether additional adjustment is needed.

[0019] In any of the above aspects, the donor organ can be a kidney, lung, heart, liver, gallbladder, pancreas, or intestine. The donor tissue can be or include a heart valve, skin tissue, bone tissue, tendon, cornea, blood vessel, cartilage tissue, ligament, eye tissue, or bone marrow tissue or other tissue. The donor tissue can include blood, platelets, cord blood stem cells, or peripheral blood stem cells. Those skilled in the art will recognize that any donor tissue or graft can be subjected to the described and claimed methods, and the above-named donor tissues are exemplary and not limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The foregoing and other objects and advantages will be apparent from the following detailed description when considered in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout, and in which:

[0021] Figure 1 shows a block diagram of a machine perfusion system for storing and / or transporting a donor organ or tissue according to an illustrative embodiment;

[0022] Figure 2 shows a flow chart depicting a method for preparing a nucleic acid preparation from a perfusion fluid according to an illustrative embodiment;

[0023] Figure 3 shows a flow chart depicting a method for predicting the outcome of transplantation by nucleic acid analysis of a perfusion fluid according to an illustrative embodiment;

[0024] Figure 4 shows a flow chart depicting a method for feedback control of mechanical perfusion of a donor organ or tissue according to an illustrative embodiment;

[0025] Figure 5A shows the DNA size distribution of cell-free DNA isolated from a perfusion fluid sample, and Figure 5B shows the DNA size distribution of cellular DNA isolated from a perfusion fluid sample;

[0026] Figure 6A shows the DNA size distribution of cell-free DNA isolated from a perfusion fluid sample, Figure 6B shows the DNA size distribution of cellular DNA isolated from a perfusion fluid sample, and Figure 6C shows the DNA size distribution of nucleosomal DNA containing a specific sample;

[0027] Figure 7 shows a graph of cellular DNA yield relative to cell-free DNA yield from a perfusion fluid sample;

[0028] Figure 8 shows a percentage chart of cell-free DNA yield relative to kidney weight for each perfusion fluid sample;

[0029] Figure 9A shows a plot of cell-free DNA yield versus perfusion time, and Figure 9B shows a plot of cellular DNA yield versus perfusion time. DETAILED DESCRIPTION

[0030] To provide an overall understanding of the systems, methods, and devices described herein, certain illustrative embodiments will be described. Although the embodiments and features described herein are specifically described in relation to the mechanical perfusion of organs and tissues, it should be understood that all of the components and other features outlined below can be combined with each other in any suitable manner and can be adapted and applied to other types of transplantation and / or perfusion methods. For example, fluid samples can be collected from static cold storage (SCS) or cryopreserved organs or tissues, or from fluids used to flush organs or tissues prior to transplantation.

[0031] The systems, devices, and methods described herein allow for the generation and subsequent analysis of nucleic acid preparations from nucleic acid samples collected from perfusion fluid (the fluid used for the mechanical perfusion of organs or tissues received from a donor) or from flush fluid (the fluid used to prepare organs or tissues prior to transplantation). While mechanical perfusion is used to preserve or recondition organs / tissues during storage or transport prior to transplantation into a recipient, according to the methods described herein, the perfusion fluid can be sampled in order to isolate and analyze nucleic acids. Similarly, the flush fluid can be sampled and analyzed during or after the preparation of organs or tissues for transplantation. Nucleic acids from the perfusion fluid, such as cell-free DNA (cfDNA), cellular DNA, or RNA, can be quantified, enriched to produce non-native preparations for analysis purposes, sequenced, amplified to produce non-native preparations for analysis purposes, or otherwise manipulated to produce non-native preparations for analysis purposes in order to inform about the current state of the organ / tissue being perfused by the physician. Analysis results, such as cfDNA concentration or fragment size distribution (or subsets thereof), can indicate that the organ / tissue has been injured and may no longer be suitable for transplantation or may require adjustment of the perfusion. Analysis results can also indicate the quality of the donor organ / tissue, which can be used to assess its suitability for transplantation and to inform the process of allocating the organ / tissue to a suitable recipient. The level of nucleic acids (such as cfDNA) in the perfusion fluid can predict primary graft dysfunction after organ / tissue transplantation.

[0032] It should be understood that the systems and methods described herein can be applied to any organ, tissue, or biological entity that is perfused or fluid - processed before or during transplantation. The present disclosure is applicable to, but not limited to, kidneys, lungs, hearts, livers, gallbladders, pancreases, intestines, heart valves, skin tissue, bone tissue, tendons, corneas, blood vessels, cartilage tissue, ligaments, eye tissue, or bone marrow tissue, or combinations thereof. In some embodiments, the organ / tissue of interest is donated blood, donated platelets, cord blood stem cells, or peripheral blood stem cells, any of which can be stored in a fluid from which a sample can be taken for nucleic acid analysis. These organs, tissues, or biological entities can be perfused and / or sampled before or after the organ is recovered from the donor. The term "graft" can be used to describe one or more of the organs or tissues described herein, and grafts applicable to these methods and systems include, but are not limited to, autografts, syngeneic grafts, allografts, and xenografts.

[0033] Although the examples in this disclosure discuss various techniques for preparing DNA preparations, DNA manipulation, and DNA analysis, it should be understood that one of ordinary skill in the art can extend this disclosure to the manipulation and analysis of RNA or any other nucleic acid present in the perfusion fluid or the flush fluid. The examples provided herein may refer only to the perfusion fluid, but it should be understood that the methods can be applied to flush fluid samples without departing from the scope of the disclosure. Both fluids (perfusion fluid and flush fluid) can be referred to herein as "graft fluid". The graft fluid can contain an oxygen carrier, a buffer or primer solution, a colloid, a nutritional supplement, an anticoagulant, a protective additive, and / or an antibiotic. The graft fluid can be a red - blood - cell - based solution, a cell solution, or whole blood. The oxygen carrier can contain red blood cells. In some embodiments, the oxygen carrier is a hemoglobin - based oxygen carrier, such as Hemopure or Breonics. Hemopure is a polymeric bovine - hemoglobin - based oxygen carrier. Breonics is a blood - free metabolic support system that contains a highly enriched tissue - culture - like medium with amino acids, lipids, carbohydrates, and bovine hemoglobin. The acellular graft solution can be It is a non - protein oxygen carrier, Aqix primarily used for preserving tissue biopsies, while the solution has a high concentration of albumin and dextran. The solution can be diluted, especially if used for kidney perfusion. The graft fluid may contain agents that prevent cellular edema, such as albumin. The graft fluid may contain agents that increase osmotic pressure and / or enhance blood flow, such as mannitol. The graft fluid may contain vasodilators, such as prostacyclin. The prostacyclin may be synthetic, such as epoprostenol. The vasodilator may also be verapamil or sodium nitroprusside. The graft fluid may contain agents that prevent inflammation, such as corticosteroids. The graft fluid may contain nutrients, such as glucose, amino acids, insulin, and / or multivitamin agents. The graft fluid may also contain antibiotics. The graft solution may contain a priming solution, such as solution, solution, plasma lysate A, or Williams media E. In a preferred embodiment, the graft fluid is a combination of solutions, may be cell-free, and may also be RBCs filled added to the perfusion fluid, optionally with autologous whole blood added to instead of RBCs. Solution composition: (1000 ml, containing 5 g of dextran 40 [0.125 mM], 70 g of bovine serum albumin [1.05 mM], 5.03 g of NaCl [85.90 mM], 0.24 g of glucose monohydrate [1.21 mM], 0.34 g of KCl [4.56 mM], 0.19 g of NaH2PO4·2H2O [1.22 mM], 0.22 g of CaCl2·2H2O [1.50 mM], 0.24 g of MgCl2·6H2O [2.52 mM], 1.26 g of NaHCO3 [15.00 mM], and adjusted to pH 7.4 with 1 M NaOH). Table 2 below includes other illustrative graft fluids disclosed in Elliott et al., Am. J. of Transpl. 21(4), 1382-1390 (2021), which is incorporated herein by reference.

[0034]

[0035]

[0036] Mechanical perfusion

[0037] Figure 1Disclosed is an exemplary machine perfusion system 100 for storing, preserving, transporting, and / or reconditioning a donor organ or donor tissue 104 for transplantation into a recipient. System 100 includes a chamber 102 for receiving the organ / tissue 104. A perfusion fluid is delivered into and out of chamber 102 via a catheter assembly 106 that couples chamber 102 to other components of system 100 such that the catheter assembly 106 and components define a fluid circuit. These components include a reservoir 108 for receiving the perfusion fluid flowing out of chamber 102. An inlet 110 is coupled to reservoir 108. These components further include a pump 112 for driving the perfusion fluid through the fluid circuit via catheter assembly 106, an oxygenator 114 for supplying oxygen to the perfusion fluid, a heat exchanger 116 for cooling or heating the perfusion fluid, and one or more sensors 118 for measuring one or more characteristics of the perfusion fluid. A separator 122 is disposed within chamber 102 (or otherwise operably coupled to chamber 102) for removing one or more substances from the chamber or perfusion fluid. A controller 120 is operably coupled to each of inlet 110, pump 112, oxygenator 114, heat exchanger 116, and sensors 118 such that controller 120 can send and / or receive data from each component.

[0038] As described in further detail below, after sampling the perfusion fluid from system 100, a method for perfusion fluid analysis can be performed. The analysis results can be used to inform feedback control of system 100 or to manually adjust certain parameters of system 100 by a doctor or operator. Although the components of system 100 are depicted in a certain order and connected in a fluid circuit via catheter assembly 106, it should be understood that these components can be rearranged in any suitable order. Two or more of the components can be arranged in series or in parallel, where in the latter configuration, the catheter assembly splits into two or more flow channels to supply the perfusion fluid through each of the two or more components. Certain components can be omitted.

[0039] It should be understood that the organ / tissue 104 can be any biological entity that requires or benefits from perfusion, e.g., for preservation, storage, transportation, and / or reconditioning. Examples include kidneys, lungs, hearts, livers, gallbladders, pancreases, intestines, heart valves, skin tissue, bone tissue, tendons, corneas, blood vessels, cartilage tissue, ligaments, eye tissue, bone marrow tissue, volume of blood, volume of platelets, volume of umbilical cord blood stem cells, or volume of peripheral blood stem cells.

[0040] The perfusion fluid delivered through the fluid circuit of system 100 can be any perfusion solution for preserving or preparing a graft. For example, the perfusion fluid can be or include a solvent, such as water, containing a combination of proteins, sugars, and / or soluble salts. In some embodiments, the perfusion fluid includes red blood cells. The perfusion fluid can be a sterile isotonic solution. For example, KPS- A kidney perfusion solution (Organ Recovery Systems, Itasca, IL) can be used for machine perfusion of the kidney. A suitable perfusion fluid can be or include a sodium lactate solution (also known as Ringer's lactate solution or Hartmann's solution). The perfusion fluid can contain at least one of sodium chloride, sodium lactate, potassium chloride, calcium chloride, magnesium sulfate, mannitol, dexamethasone, glutathione, or insulin. In any embodiment, the perfusion fluid within chamber 102 and the perfusion fluid delivered out of chamber 102 through catheter assembly 106 contain nucleic acids from organ / tissue 104. The nucleic acids in the perfusion fluid can be in the form of cellular DNA, cell-free DNA, or RNA, or a combination thereof.

[0041] System 100 can include a housing such that one or more components are enclosed within the housing. For example, each component can be enclosed within the housing. The housing can include one or more ports for connection to an external source or drain, for example, for inlet 110 or separator 122. The housing can be a transporter configured with a carrying handle for transporting and storing donor organ / tissue 104. Alternatively, system 100 does not include a housing. The components can be loosely arranged and connected via the wires of catheter assembly 106 and controller 120. In some embodiments, chamber 102 is at least a part of a cadaver and the organ / tissue 104 has not been extracted from the cadaver. The remainder of system 100 can be enclosed within a housing that is fluidly connected to the cadaver via catheter assembly 106.

[0042] Chamber 102 is a container for aseptically containing organ / tissue 104 during storage or transportation and is generally used for the preservation and / or reconditioning of organ / tissue 104. Chamber 102 can be a container having walls, an inlet, and an outlet, the inlet and outlet being fluidly coupled to catheter assembly 106 for delivering perfusion fluid into and out of chamber 102. Chamber 102 can be a sterile and / or disposable container. Chamber 102 can be constructed from a biocompatible material such as thermoplastic elastomer (TPE). Chamber 102 can include a lid that allows access to chamber 102 and organ / tissue 104. Chamber 102 can have a rigid, flexible, or collapsible structure. In some embodiments, chamber 102 includes one or more measurement ports and sensor 118 can be inserted into chamber 102 via one or more measurement ports for measuring characteristics of the perfusion fluid, characteristics of organ / tissue 104, and / or conditions of chamber 102. Examples of measurements will be discussed in further detail below.

[0043] The catheter assembly 106 can be formed from any suitable tubing material, such as a biocompatible plastic. The catheter assembly 106 can be rigid or flexible. In some embodiments, wires are attached to or formed on the catheter assembly 106 for connection between the controller 120 and any one of the pump 112, the oxygenator 114, the heat exchanger 116, or the sensor 118.

[0044] The reservoir 108 receives the perfusate delivered from the chamber 102 via the catheter assembly 106. The reservoir 108 can be a containment chamber having a perfusate inlet and a perfusate outlet, each coupled to the catheter assembly 106 for delivering the perfusate. The inlet 110 is coupled to the reservoir 108 for introducing substances, such as fresh perfusate, fresh perfusate components, therapeutic agents, nutritional supplies, stem cells, and / or blood cells. The inlet 110 can be operatively coupled to the controller 120 such that substances can be added to the perfusate in response to user input to the controller 120 or in a feedback control loop implemented by the controller 120, as discussed further in detail below. In some embodiments, a separator 122 is connected to or disposed in the reservoir 108 and removes certain substances from the perfusate, as discussed further in detail below.

[0045] The pump 112 drives the perfusate through the fluid circuit of the system 100. The pump 112 can be any suitable type of fluid pump, such as a positive displacement pump (e.g., a peristaltic pump, a plunger pump, or a piston pump) or a non-positive displacement pump (e.g., a centrifugal pump). The pump 112 can induce a continuous or pulsatile flow of the perfusate. The pump 112 is operatively coupled to the controller 120, which can set the flow rate of the pump 112 using user input or using feedback control. The flow rate can be set based on per mass of the organ / tissue 104.

[0046] The oxygenator 114 supplies oxygen to the perfusate. The oxygenator 114 can include a port for receiving oxygen from an external source, such as an oxygen concentrator, an oxygen tank, or a wall oxygen supply. Alternatively, the oxygenator 114 can include an oxygen tank or an oxygen concentrator. For example, the oxygenator 114 is an oxygen concentrator and includes an inlet for receiving ambient air, which is then concentrated to produce an oxygen-rich air stream for oxygenation of the perfusate.

[0047] The heat exchanger 116 is configured to cool or heat the perfusion fluid. The perfusion of the organ / tissue 104 using the system 100 can be performed at different temperatures (ranging from hypothermic (4 °C to 10 °C) to sub-normal (15 °C to 30 °C) and normal (about 37 °C)). Thus, the heat exchanger 116 can be used to maintain the target temperature of the perfusion fluid (e.g., between 0 °C and 40 °C) by heating or cooling. The controller 120 is operably coupled to the heat exchanger 116 to set the target temperature and / or adjust the temperature of the perfusion fluid. The controller 120 can accept user input to set or adjust the target temperature and then control the heat exchanger 116 to reach the target temperature. In some embodiments, a feedback control method is used to adjust the temperature of the perfusion fluid. The heat exchanger 116 may include a temperature sensor, or the sensor 118 can measure the temperature and transmit the measured temperature to the controller 120 to appropriately control the heat exchanger 116.

[0048] The sensor 118 is configured to measure one or more characteristics of the perfusion fluid. The characteristics measured by the sensor 118 can include at least one of temperature, flow rate, pH, oxygen concentration, nucleic acid concentration, toxin concentration, or the concentration of another perfusion fluid component. The value of the measured characteristic is transmitted by the sensor 118 to the controller 120. The measured values can be used for feedback control of one or more components of the system 100 or for informing clinical decisions.

[0049] The separator 122 can be used to remove substances from the chamber 102. Although shown adjacent to or attached to the chamber 102 in Figure 1 , it should be understood that the separator or outlet can be provided at any point in the fluid circuit of the system 100, e.g., in the catheter assembly 106 or in the reservoir 108. For example, the separator 122 is a filter (e.g., a microporous filter, an ultrafiltration device), a separation column, a sediment trap, or a probe set (e.g., an affinity probe, a magnetic probe). Substances removed by the separator 122 (e.g., metabolites) can be flushed from the system 100.

[0050] The controller 120 is, for example, a microcontroller, a processor, or a printed circuit board, configured to receive data or user input and control various components of the system 100. The controller 120 can implement adaptive feedback control of the perfusion based on the measured perfusion characteristics or by receiving input of the stability or damage characteristics of the organ / tissue 104. Such input can be the result generated according to the analysis methods described herein.

[0051] Perfusion fluid sample preparation method

[0052] As described above, perfusion fluid from a mechanical perfusion system (such as system 100) can be sampled, and nucleic acids in the perfusion fluid sample can be manipulated, amplified, prepared, and / or analyzed according to the methods described herein.Figures 2 to 4 Methods for performing and utilizing such nucleic acid analysis are described.

[0053] Figure 2 A flowchart depicting an exemplary method 200 is shown, which is for preparing a nucleic acid sample from a perfusion fluid used for preserving and / or reconditioning a donor organ / tissue. This method is particularly useful for evaluating or predicting the quality of a donor organ / tissue transplantation and the subsequent transplantation outcome of the donor organ / tissue. Method 200 includes steps 202, 204, and 206. Step 202 involves obtaining a sample of the perfusion fluid from the donor organ / tissue or a machine perfusion system (such as system 100 described above with respect to Figure 1 The sample includes nucleic acids. Step 204 involves separating the nucleic acids from the sample. Step 206 involves analyzing the isolated DNA preparation to evaluate at least one of the following: the amount of nucleic acids in the perfusion fluid, the molecular weight of the nucleic acids in the perfusion fluid, or the fragment size distribution of the nucleic acids in the perfusion fluid.

[0054] The nucleic acids separated from the sample in step 204 can be cellular DNA, cell-free DNA (cfDNA), or RNA. Thus, separation techniques (including but not limited to centrifugation, size selection, hybridization capture, or other suitable techniques) can be used to separate cellular DNA, cfDNA, or RNA from the perfusion fluid in the sample. In some embodiments, the sample can be centrifuged to separate the layers. In some embodiments, filtration can be used to separate the nucleic acids. In some embodiments, the preparation of the nucleic acids can involve amplification, separation, separation by chromatography, liquid-liquid separation, preferential enrichment, preferential amplification, targeted amplification, reverse transcription, or any one of many other techniques described herein, or any combination thereof. In some embodiments of separating DNA, RNase is used to degrade RNA. Alternatively, deoxyribonuclease can be used to degrade DNA to isolate RNA. Suitable purification techniques further include but are not limited to: differential ultracentrifugation, density gradient ultracentrifugation, polymer-promoted precipitation, immunoaffinity capture, and size exclusion chromatography. Those of ordinary skill in the art will recognize that the foregoing separation techniques are exemplary and not exhaustive. The foregoing or other separation techniques can be combined with measurement, quantification, and multi-omics characterization techniques on a microfluidic platform.

[0055] For example, centrifugation can be used to separate cell clumps from the perfusion fluid. Methods or devices such as (but not limited to) cell DNA-specific probes, size selection, or silicon-based membranes with selective binding can be used to purify cell DNA from the cell clumps, and cfDNA can be purified from the remaining perfusion fluid by methods or devices such as (but not limited to) cfDNA-specific probes, size selection, or circular DNA systems. Nucleic acids can be isolated from cell sources by a variety of extraction methods. Such methods can involve lysing the cells to release the nucleic acids, leaving the chromatin structure intact enough to allow preparation of a nucleosome ladder, i.e., nucleosome preparation. Suitable cell lysis methods include methods that separately release the cell nuclei for subsequent isolation and methods that dissolve the nuclear membrane. In some embodiments, the cells can be permeabilized, for example using a detergent such as lysophosphatidylcholine, to maintain the chromatin structure. In some embodiments, in the cell source cells, the cell membrane can be disrupted by inducing apoptosis. Preparation of nucleic acids free of other cell components is of interest to enable biochemical manipulation of the nucleosome ladder for use in subsequent procedures such as DNA sequencing. In an embodiment, a nucleic acid system can be used to purify cell DNA. For example, beads with special surface chemistry can be used to isolate cfDNA from the remaining perfusion fluid.

[0056] Techniques such as amplification can be used between steps 204 and 206 to further prepare, modify, purify, and / or enrich the isolated nucleic acid. In some embodiments, universal-labeled adapters are added to generate a library. In some embodiments, PCR can be used to add labeled adapters. In some embodiments, ligation can be used to add labeled adapters. Prior to ligation, the sample nucleic acid can be blunt-ended and then a single adenosine base can be added to the 3' end. Prior to ligation, the nucleic acid can be cleaved using a restriction enzyme or some other cleavage method. During ligation, the 3' adenosine of the sample fragment and the complementary 3' tyrosine overhang of the adapter can enhance ligation efficiency. In some embodiments, universal primers are used to amplify the library. In embodiments, the amplified library is fractionated by size separation or other methods. In some embodiments, PCR amplification is used to amplify the target locus. In some embodiments, the amplified nucleic acid is sequenced (such as, for example, using an ILLUMINA IIGAX or HiSeq sequencer). In some embodiments, the amplified nucleic acid is sequenced from each end of the amplified nucleic acid to reduce sequencing errors. If there is a sequence error in a particular base when sequencing from one end of the amplified nucleic acid, it is less likely that there will be a sequence error in the complementary base when sequencing from the other side of the amplified nucleic acid (compared to sequencing multiple times from the same end of the amplified nucleic acid). Thus, the amplified nucleic acid can be re-sequenced (or the number of sequencing cycles can be increased) from one or both ends to increase the "read depth". As used herein, the term "read depth" refers to the number of sequencing reads mapped to a given locus. The read depth can be normalized against the total number of reads. When "read depth" refers to a sample, this can mean the average read depth of the targeted locus. When "read depth" refers to a locus, it can refer to the number of reads measured by the sequencer that are mapped to that locus. Generally, the greater the read depth of a locus, the more likely the ratio of alleles at the locus is to approximate the ratio of alleles in the original DNA sample. Typically, increasing the read depth can reduce errors in sequencing.

[0057] In some embodiments, whole genome amplification (WGA) is used to amplify a nucleic acid sample. In one embodiment, WGA is performed using ligation-mediated PCR (LM-PCR), where short DNA sequences called adapters are ligated to blunt ends of DNA. These adapters contain universal amplification sequences that are used to amplify the DNA by PCR. In another embodiment, WGA is performed using degenerate oligonucleotide primer PCR (DOP-PCR), where random primers that also contain universal amplification sequences are used in the first round of annealing and PCR. Then, a second round of PCR is used to further amplify the sequences with universal primer sequences. In another embodiment, WGA is performed using multiple displacement amplification (MDA), which uses phi-29 polymerase, a highly processive and non-specific enzyme that replicates DNA and has been used for single cell analysis. In some embodiments, WGA is not performed.

[0058] In some embodiments, selective amplification or enrichment is used to amplify or enrich target loci. In some embodiments, amplification and / or selective enrichment techniques can involve PCR (such as ligation-mediated PCR), fragment capture by hybridization, molecular inversion probes, or other in-circle probes. In some embodiments, real-time quantitative PCR (RT-qPCR), digital PCR, droplet PCR, or emulsion PCR, single allele base extension reactions followed by mass spectrometry (Hung et al., J Clin Pathol 62:308-313, 2009, which is hereby incorporated by reference in its entirety) are used. In some embodiments, capture by hybridization with hybridization capture probes is used to preferentially enrich nucleic acids. In some embodiments, methods for amplification or selective enrichment can involve the use of probes, where after correct hybridization to the target sequence, the 3' or 5' end of the nucleotide probe is split from the polymorphic site of the polymorphic allele by a small number of nucleotides. This splitting reduces the preferential amplification of one allele, called allelic bias. This is an improvement over methods that involve the use of probes where the 3' or 5' end of the correctly hybridized probe is directly adjacent or very close to the polymorphic site of the allele. In one embodiment, probes where the hybridization region may or is determined to contain a polymorphic site are excluded. The polymorphic site at the hybridization site can cause unequal hybridization of some alleles or inhibit overall hybridization, resulting in preferential amplification of certain alleles. The advantage of these embodiments over other methods involving targeted amplification and / or selective enrichment is that these embodiments better maintain the original allelic frequencies of the sample at each polymorphic locus, whether the sample is a pure genomic sample from a single individual or a mixture of individuals.

[0059] In some embodiments, a PCR technique called miniPCR is used to generate very short amplicons (U.S. Application Serial No. 13 / 683,604, filed November 21, 2012; U.S. Publication No. 2013 / 0123120; U.S. Application Serial No. 13 / 300,235, filed November 18, 2011; U.S. Publication No. 2012 / 0270212, filed November 18, 2011; and U.S. Serial No. 61 / 994,791, filed May 16, 2014, each of which is hereby incorporated by reference in its entirety). cfDNA is highly fragmented. For some cfDNA, the fragment sizes are distributed approximately in a Gaussian manner, with a mean of 160 bp, a standard deviation of 15 bp, a minimum size of about 100 bp, and a maximum size of about 220 bp. The polymorphic site of a particular target locus can occupy any position from start to end in the various fragments derived from this locus. Because the cfDNA fragments are short, the likelihood of the presence of two primer sites, including the likelihood of a fragment of length L with both forward and reverse primer sites, is the ratio of the amplicon length to the fragment length. Under ideal conditions, assays where the amplicon is 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, or 70 bp will successfully amplify from 72%, 69%, 66%, 63%, 59%, or 56% of the available template fragment molecules, respectively. In certain embodiments, cfDNA is amplified using primers that produce amplicons of 85 bp, 80 bp, 75 bp, or 70 bp and in certain preferred embodiments, a maximum amplicon length of 75 bp and having a melting temperature between 50 °C and 65 °C and in certain preferred embodiments, between 54 °C - 60.5 °C. The amplicon length is the distance between the 5' ends of the forward and reverse primer sites. Shorter amplicon lengths than typically used can enable more efficient measurement of the desired polymorphic locus by requiring only short sequence reads. In one embodiment, a substantial portion of the amplicon is less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp.

[0060] In some embodiments, the amplification is performed using direct multiplex PCR, sequence PCR, nested PCR, double nested PCR, semi-nested PCR, complete nested PCR, single-sided complete nested PCR, single-sided nested PCR, semi-nested PCR, semi-nested PCR, triple semi-nested PCR, semi-nested PCR, single-sided semi-nested PCR, inverse semi-nested PCR, or single-sided PCR, which are described in the following: U.S. Application Serial No. 13 / 683,604 filed on November 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application Serial No. 13 / 300,235 filed on November 18, 2011, U.S. Publication No. 2012 / 0270212, and U.S. Serial No. 61 / 994,791 filed on May 16, 2014, which are hereby incorporated by reference in their entirety. Optionally, any of these methods can be used for mini-PCR.

[0061] Optionally, the extension step of the PCR amplification can be limited from a time perspective to reduce amplification from fragments longer than 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, or 1,000 nucleotides. This can result in fragmentation or enrichment of shorter DNA (such as DNA from cells undergoing apoptosis or necrosis) and improved test performance.

[0062] In some embodiments, multiplex PCR is used. In some embodiments, a method of amplifying a target locus in a nucleic acid sample involves (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 100; 200; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci to produce a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to produce an amplification product containing the target amplicons. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the targeted loci are amplified. In various embodiments, less than 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, or 0.05% of the amplification products are primer dimers. In some embodiments, the primers are in solution (such as dissolved in the liquid phase rather than the solid phase). In some embodiments, the primers are in solution and are not immobilized on a solid support. In some embodiments, the primers are not part of a microarray. In some embodiments, the primers do not include molecular inversion probes (MIPs).

[0063] Step 206 involves analyzing the isolated nucleic acid preparation to assess at least one of the following: the amount of nucleic acid in the perfusion fluid, the molecular weight of the nucleic acid in the perfusion fluid, or the fragment size distribution of the nucleic acid in the perfusion fluid. A variety of techniques can be used to perform this analysis. The appropriate technique can be selected by balancing variations in accuracy, reproducibility, sensitivity, labor intensity, speed, and cost among the various techniques. Suitable techniques include, for example, using NANODROP 1000 (THERMO SCIENTIFIC), QUBIT 2.0 High Sensitivity Assay (LIFE TECHNOLOGIES), BIOANALYZER 2100 High Sensitivity Assay (AGILENT TECHNOLOGIES), TAPESTATION 2200 High Sensitivity D1000 Kit (AGILENT TECHNOLOGIES), GXTOUCH 24 High Sensitivity Assay (PERKINELMER), or FRAGMENT ANALYZER High Sensitivity NGS Kit (ADVANCED ANALYTICAL). Suitable methods / techniques include, but are not limited to, ultraviolet-visible spectrophotometry, fluorescence methods (e.g., fluorescence dye-based methods such as fluorescence in situ hybridization (FISH)), microfluidics, electrophoresis, automated electrophoresis, capillary electrophoresis, and droplet-based methods. In some embodiments, qPCR is used to measure cellular DNA, cfDNA, or RNA. For example, multiplex qPCR can be used to measure one or more loci from cfDNA or cellular DNA (such as glyceraldehyde-3-phosphate dehydrogenase, GAPDH). In some embodiments, fluorescence-labeled PCR is used to measure cfDNA, cellular DNA, or RNA. As needed, methods such as, but not limited to, the Shapiro-Wilk-Test can be used to calculate the normal distribution of the data. As needed, methods such as the Mann-Whitney-U-Test can be used to compare cfDNA, cellular DNA, or RNA levels. In some embodiments, methods such as the Mann-Whitney U test or the Kruskal-Wallis-Test are used to compare cfDNA, cellular DNA, or RNA levels with other prognostic factors.

[0064] The amount of nucleic acid evaluated in step 206 can be the total amount of nucleic acid in the perfusion fluid sample, an estimate of the total amount of nucleic acid in the perfusion fluid of the machine perfusion system, the concentration of nucleic acid in the perfusion fluid sample, the amount of low molecular weight DNA in the sample (e.g., fragments less than 200 base pairs), or the amount of high molecular weight DNA in the sample (e.g., fragments greater than or equal to 200 base pairs). The molecular weight of the nucleic acid evaluated in step 206 can be the average molecular weight of the nucleic acid in the perfusion fluid or the molecular weight distribution of the nucleic acid.

[0065] Method 200 can include additional steps for interpreting or improving the analysis results of step 206. For example, when the nucleic acid isolated and analyzed is cfDNA, if the molecular weight of the cfDNA (evaluated in step 206) is consistent with nucleosomal DNA, method 200 can further evaluate that the cfDNA is derived from apoptosis. The method can further involve evaluating changes in homeostasis or cellular processes based on the analysis results. For example, an elevated level of cfDNA in the perfusion fluid can indicate that the organ / tissue has lost homeostasis and that failure may be occurring or has occurred.

[0066] In some embodiments, the isolated nucleic acid in the preparation is RNA. The RNA can be RNA present in extracellular vesicles such as exosomes and microvesicles. Vesicles captured from the perfusion fluid can be lysed to release the RNA, which can then be purified in solution using techniques such as filtration, size selection, or hybridization capture. The method can further include evaluating the amount of isolated RNA as a biomarker of organ / tissue stability or quality. The method can further include evaluating the gene expression profile corresponding to the isolated RNA to assess the cellular origin of the RNA. For example, when the gene expression profile is related to immune function, the cellular origin of the RNA is evaluated as immune cells. RNA can be used as a biomarker like DNA, but for example, when packaged in vesicles, it can also provide a better source image (i.e., better characterize cell viability or organ status) compared to free RNA. Due to the role of RNA in gene expression, the analysis of RNA can provide more functional information about the organ or tissue state compared to the analysis of DNA. Due to the instability of RNA (due to its single-stranded form, uracil degradation, and the ubiquitous ribonucleases in cells and tissues, which can rapidly degrade RNA) and the lower natural RNA concentration, the manipulation and analysis of RNA may be more difficult than that of DNA. Therefore, additional techniques may be required to accurately isolate and analyze RNA from perfusion fluid or lavage fluid samples. For example, more selective isolation and extraction methods can be used to account for the lower RNA concentration. To improve stability, RNA can be reverse transcribed to produce complementary DNA (cDNA).

[0067] To address the instability and low concentration of RNA, suitable techniques for isolating RNA from a sample or preparation include (but are not limited to) guanidinium phenol extraction, filter techniques (e.g., glass fiber filters), density gradient centrifugation (e.g., using cesium chloride or cesium trifluoroacetate), magnetic bead techniques (e.g., hybridization capture using biotinylated probes and streptavidin-coated magnetic beads), lithium chloride and urea separation, chromatography (e.g., oligo(dt)-cellulose column chromatography), and non-column poly(A)+ purification / separation. Suitable techniques can involve cell lysis and solubilization, denaturation of DNA and proteins, denaturation and inactivation of RNases, removal or separation or precipitation of cellular components. In some embodiments, the RNA is reverse transcribed to produce cDNA, which is then analyzed in step 206. The results traceable to the cDNA analysis are used to characterize the RNA in the original sample or preparation.

[0068] In some embodiments, RNA (e.g., mRNA) is analyzed from the perfusate or wash fluid to evaluate the cellular transcriptome of the graft at any given time. Thus, the method can involve transcriptome assembly from sequencing reads of the RNA or corresponding cDNA (e.g., by microarray or RNA-Seq). In the case of cDNA, suitable sequencing techniques include but are not limited to next-generation sequencing (high-throughput sequencing), shotgun sequencing, Sanger sequencing, pyrosequencing, or nanopore sequencing. Transcriptome assembly can be de novo (without a reference genome), so that pre-genotyping of the graft or donor is not required, nor is a genomic guide. The de novo method involves identifying adjacent sequences in the sequence reads (e.g., by using a de Bruijn graph). When the genome of the graft or donor is already known, a genome-guided method can be used. Alternatively, DNA in the same or another perfusate or wash fluid sample can be genotyped in parallel with the RNA analysis to generate a reference genome. The genome-guided method aligns the sequence reads of both adjacent and non-adjacent sequences.

[0069] RNA analysis can also involve quantification of gene expression. Quantification of expression can be used to study changes in response to external stimuli, differences between healthy and diseased states, and other issues related to graft stability. For example, changes in perfusion (or wash) conditions (e.g., temperature, pH, solute concentration) can result in changes in gene expression within the graft cells, and analysis of this dynamic expression can be used to inform adjustments to the perfusion (or wash) conditions, such as as described below with respect to Figure 4As described above, expression can be quantified by counting the number of reads mapped to each genetic locus in the transcriptome assembly step. Expression of exons or genes can be quantified, for example, using identified contiguous or reference transcript annotations. Observed read counts can be converted to appropriate metrics for hypothesis testing, regression, or other analyses, such as considering sequence depth or coverage, gene length, total sample RNA (e.g., evaluated in step 206), or differences in expression of each gene. Considering these parameters can help normalize results between samples and loci and reduce errors (e.g., sampling errors propagated through the analysis).

[0070] As described herein, multiple samples (of perfusate or wash fluid) can be collected over time or for different grafts and processed according to method 200. RNA analysis of multiple samples can be particularly useful for assessing differential expression, where expression varies between two or more conditions (e.g., perfusion conditions such as temperature or pH) or two or more candidate grafts (e.g., from the same donor or cadaver, or from donors with the same or similar genomes). One or more samples are collected for each condition or candidate graft. The output of differential expression analysis performed on multiple samples includes differentially expressed genes (DEGs), which can be upregulated or downregulated. Differential expression analysis can take as input: (1) a gene expression matrix including M genes in each of N samples via gene expression quantification analysis; and (2) a design matrix of the experimental conditions for the N samples. Relevant conditions include (but are not limited to) physical conditions (e.g., temperature, pH, or concentration of perfusate or wash fluid; graft size), batch effects (e.g., laboratory conditions, measurement error, instruments or techniques used, reagent lot or batch, personnel differences, time of day), genetic effects (e.g., known relics or variants), and / or any metadata that can alter gene expression. Conditions can be known or unknown, where unknown conditions can be estimated via machine learning methods (e.g., principal components, surrogate variables). Hidden variable analysis can also be performed to identify conditions not yet captured. Differential expression analysis can methodologically involve regression or nonparametric statistics to identify DEGs. Adjustment methods (such as family-wise error rate or false discovery rate) can be employed to account for multiple hypotheses. The output of differential expression analysis can include a table showing the log fold change, p-value, and / or adjusted p-value for multiple comparisons for each gene. A log fold change cut-off can be set to identify biologically relevant DEGs (DEGs that pass the cut-off and are statistically significant). The identified biologically relevant DEGs can be used to inform decisions about the graft. For example, if immune response-related genes are upregulated, the graft may require perfusion adjustment or treatment with a drug (e.g., an immunostabilizer). Certain genes can be identified as biomarkers of graft rejection or failure, and thus upregulation of these genes can be an indicator of predicted graft rejection or failure.

[0071] The result of step 206 can be normalized to account for certain parameters of the machine perfusion system from which the sample was taken. For example, the amount of nucleic acid, molecular weight, or fragment size distribution is normalized relative to one or more of perfusion time (the period of time the organ / tissue has been perfused), perfusion fluid volume, organ / tissue size, organ / tissue weight, organ / tissue volume, organ / tissue surface area, perfusion fluid temperature, and perfusion fluid pH value. Thus, each of these parameters can be measured before or during method 200.

[0072] Method 200 can further include the step of assessing the fragmentation pattern of the nucleic acid. This fragmentation pattern can then be used to classify the isolated nucleic acid as nucleic acid randomly degraded from lysed cells in the perfusion fluid and nucleic acid from apoptosis of cells in the donor organ / tissue.

[0073] Additional samples of the perfusion fluid can be taken from the donor organ / tissue at different time points. By repeating steps 204 and 206 on the additional samples, analytical results such as the amount of nucleic acid or the molecular weight of the nucleic acid can be tracked over a period of time. By monitoring the amount of nucleic acid, molecular weight, or fragment size distribution (or a subset thereof) over time, a doctor may be able to closely monitor the quality or status of the donor organ / tissue and assess whether intervention or adjustment is needed, as discussed in further detail below.

[0074] Figure 3 A flowchart depicting method 300 for predicting the outcome of a transplantation is shown. Method 300 includes steps 302, 304, 306, and 308. Step 302 involves obtaining a sample of the perfusion fluid from a donor organ / tissue or a machine perfusion system (such as system 100 described above with respect to Figure 1 ). The sample includes nucleic acid. Step 304 involves isolating the nucleic acid from the sample to produce an artificial preparation of the nucleic acid. Step 306 involves analyzing the preparation of the isolated nucleic acid to assess at least one of the following: the amount of nucleic acid in the perfusion fluid, the molecular weight of the nucleic acid in the perfusion fluid, or the fragment size distribution of the nucleic acid in the perfusion fluid. Steps 302, 304, and 306 can be performed in the same manner as steps 202, 204, and 206 described above with respect to Figure 2 . Step 308 involves predicting the transplantation outcome based on the analysis performed in step 306, i.e., based on at least one of the amount of nucleic acid, the molecular weight of the nucleic acid, or the fragment size distribution (or a subset thereof) of the nucleic acid.

[0075] The predicted outcomes can include the presence of delayed graft function, the duration of delayed graft function, the primary non - function rate, organ function at various time points after transplantation, the likelihood of transplant rejection, the likelihood of non - rejection of the transplant, the type of non - rejection of the transplant, or the time of rejection.

[0076] The analysis result of step 306 can provide information on the quality or status of the donor organ or tissue, or provide information on the clinical outcome of transplanting the organ or tissue into the recipient. For example, when the isolated and analyzed nucleic acid preparation is cellular DNA from cells of a donor source, the predicted result of step 308 can be a favorable or unfavorable prognosis predicted based on the cellular DNA, which indicates the immune response of the cells of the donor source of the organ / tissue. As another example, the isolated and analyzed nucleic acid is cfDNA, and the predicted result is a favorable or unfavorable prognosis predicted based on the cfDNA, which indicates damage to the donor organ / tissue. The predicted result of step 308 can be used as a decision maker for adjusting perfusion parameters or not using the organ / tissue for transplantation. For example, if the predicted result is a predicted favorable or unfavorable prognosis. The predicted result can be used to recommend adjusting or stopping the perfusion of the organ / tissue. For example, when the evaluated amount of nucleic acid in the perfusion fluid exceeds the threshold amount of nucleic acid, it is recommended to stop perfusion.

[0077] The result of step 308 can be predicted based at least in part on a risk determination generated by an algorithm that uses the evaluated amount, molecular weight, or fragment size distribution (or a subset thereof) of the nucleic acid as algorithm inputs. The result can be predicted by quality assessment of the donor organ / tissue based on the amount, molecular weight, or fragment size distribution (or a subset thereof) of the nucleic acid. Various methods can be used for quality assessment. For example, a modified kidney donor risk index can be calculated by combining the evaluated amount, molecular weight, or fragment size distribution (or a subset thereof) of the nucleic acid in order to summarize the risk of kidney graft failure. Quality assessment can involve scoring the graft based on nucleic acid analysis. The scoring includes, but is not limited to, calculating a graft-specific quality index (e.g., liver graft quality index, lung graft quality index, or kidney donor risk index (KDPI)).

[0078] Figure 4 A flowchart depicting a method 400 for feedback control of machine perfusion of a donor organ or tissue is shown. Method 400 includes steps 402, 404, 406, 408, and 410. Step 402 involves preserving the donor organ or tissue by machine perfusion at a first value of perfusion parameters. Step 404 involves obtaining a sample of the perfusion fluid from the donor organ / tissue or the machine perfusion system used for preservation in step 402 (such as system 100 described above with respect to Figure 1 described system 100). The sample includes nucleic acids. Step 406 involves isolating the nucleic acids from the sample. Step 408 involves analyzing the isolated nucleic acids to evaluate at least one of the following: the amount of nucleic acid in the perfusion fluid, the molecular weight of the nucleic acid in the perfusion fluid, or the fragment size distribution of the nucleic acid in the perfusion fluid. Steps 404, 406, and 408 can be in accordance with those described above with respect to Figure 2 and Figure 3performed according to any of the embodiments described in steps 202 / 302, 204 / 304, and 206 / 306. Step 410 involves appropriately adjusting the perfusion parameters of the machine perfusion based at least in part on the analysis results (e.g., based on at least one of the amount of nucleic acid, molecular weight, or fragment size distribution (or a subset thereof)). Step 412 involves adjusting the perfusion parameters of the machine perfusion to a second value based on the generated appropriate adjustment.

[0079] Steps 410 and 412 can be performed in an automated manner by a controller (such as the controller 120 described above with respect to Figure 1 the controller 120). The controller can include machine-readable instructions for performing feedback control of the perfusion parameters. Multiple perfusion parameters can be controlled in a coordinated feedback manner. The controller can use, but is not limited to, proportional control, proportional-integral control, or proportional-integral-derivative (PID) control. The feedback control can respond to user input of the analysis results of step 408. Alternatively, the machine perfusion can be performed on a system that allows the user to control the perfusion parameters, and the adjustment of the perfusion parameters can be performed manually by a doctor / user.

[0080] The perfusion parameters can include, but are not limited to: perfusion fluid flow rate, perfusion fluid temperature, perfusion fluid pH value, oxygen concentration of the perfusion fluid, concentration of therapeutic drugs (e.g., immunosuppressive or anti-inflammatory drugs, stem cells), concentration of perfusion fluid components (e.g., salts, proteins, nutrients), or perfusion duration. The appropriate adjustment can include a recommendation to adjust more than one perfusion parameter simultaneously or sequentially.

[0081] In some embodiments, method 400 further includes the step of collecting an adjusted perfusion fluid sample and evaluating whether additional adjustment is needed. Any number of iterations of the adjustment step and re-evaluation of additional adjustment can be performed.

[0082] Other techniques

[0083] Methods 200, 300, and / or 400 can further include, at any point after separating the nucleic acid from the perfusion fluid sample, performing targeted genetic analysis on the separated nucleic acid to identify one or more genetic characteristics in the nucleic acid. Such targeted genetic analysis can be performed using amplification and sequencing (e.g., high-throughput sequencing, microarray, nanopore sequencing).

[0084] In some embodiments, the method includes separating or purifying nucleic acids. There are many procedures to achieve this goal. In some embodiments, the sample can be centrifuged to split the layers. In some embodiments, filtration can be used to separate nucleic acids. In some embodiments, the preparation of nucleic acids can involve amplification, separation, purification by chromatography, liquid-liquid separation, isolation, preferential enrichment, preferential amplification, targeted amplification, reverse transcription, or any combination thereof, or any one of many other techniques described herein. In some embodiments of separating DNA, ribonuclease is used to degrade RNA. Alternatively, deoxyribonuclease can be used to degrade DNA to isolate RNA in the sample.

[0085] In some embodiments, universal-tagged adapters are added to generate a library. Prior to ligation, the sample nucleic acids can be blunt-ended and then a single adenosine base can be added to the 3' end. In some embodiments, PCR can be used to add tagged adapters. In some embodiments, ligation can be used to add tagged adapters. Prior to ligation, restriction enzymes or some other cleavage method can be used to cleave the nucleic acids. During ligation, the 3' adenosine of the sample fragment and the complementary 3' tyrosine overhang of the adapter can enhance ligation efficiency. In some embodiments, universal primers are used to amplify the library. In embodiments, the amplified library is fractionated by size separation or other methods. In some embodiments, PCR amplification is used to amplify target loci. In some embodiments, the amplified nucleic acids are sequenced (such as using an ILLUMINA IIGAX or HiSeq sequencer). In some embodiments, the amplified nucleic acids are sequenced from each end of the amplified nucleic acids to reduce sequencing errors. If there is a sequence error in a particular base when sequencing from one end of the amplified nucleic acids, it is less likely that there will be a sequence error in the complementary base when sequencing from the other side of the amplified nucleic acids (compared to sequencing multiple times from the same end of the amplified nucleic acids).

[0086] In some embodiments, whole genome amplification (WGA) is used to amplify a nucleic acid sample. There are multiple methods available for WGA: ligase-mediated PCR (LM-PCR) for WGA, where short DNA sequences called adapters are ligated to blunt ends of DNA. These adapters contain universal amplification sequences, which are used to amplify DNA by PCR. In another embodiment, WGA is performed using degenerate oligonucleotide primer PCR (DOP-PCR), where random primers that also contain universal amplification sequences are used in the first round of annealing and PCR. Then, a second round of PCR is used to further amplify the sequences with universal primer sequences. In another embodiment, WGA is performed using multiple displacement amplification (MDA), which uses phi-29 polymerase, a highly processive and non-specific enzyme that replicates nucleic acids and has been used for single cell analysis. In some embodiments, WGA is not performed.

[0087] In some embodiments, selective amplification or enrichment is used to amplify or enrich target loci. In some embodiments, amplification and / or selective enrichment techniques can involve PCR (such as ligase-mediated PCR), fragment capture by hybridization, molecular inversion probes, or other in-loop probes. In some embodiments, real-time quantitative PCR (RT-qPCR), digital PCR, droplet PCR, or emulsion PCR, single allele base extension reactions followed by mass spectrometry (Hung et al., J Clin Pathol 62:308-313, 2009, which is hereby incorporated by reference in its entirety) are used. In some embodiments, capture by hybridization using hybridization capture probes is used to preferentially enrich nucleic acids. In some embodiments, methods for amplification or selective enrichment can involve using probes, where after correct hybridization to the target sequence, the 3' or 5' end of the nucleotide probe is split from the polymorphic site of the polymorphic allele by a small number of nucleotides. This splitting reduces the preferential amplification of one allele, called allelic bias. This is an improvement over methods that involve using probes where the 3' or 5' end of the correctly hybridized probe is directly adjacent or very close to the polymorphic site of the allele. In one embodiment, probes where the hybridization region can or is determined to contain a polymorphic site are excluded. Polymorphic sites at the hybridization site can cause unequal hybridization of some alleles or inhibit overall hybridization, resulting in preferential amplification of certain alleles. The advantage of these embodiments over other methods involving targeted amplification and / or selective enrichment is that these embodiments better maintain the original allelic frequencies of the sample at each polymorphic locus, whether the sample is a pure genomic sample from a single individual or a mixture of individuals.

[0088] In some embodiments, a PCR technique called miniPCR is used to generate very short amplicons (U.S. Application Serial No. 13 / 683,604, filed November 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application Serial No. 13 / 300,235, filed November 18, 2011, U.S. Publication No. 2012 / 0270212, filed November 18, 2011, and U.S. Serial No. 61 / 994,791, filed May 16, 2014, each of which is hereby incorporated by reference in its entirety). cfDNA is highly fragmented. For some cfDNA, the fragment sizes are distributed approximately in a Gaussian manner, with a mean of 160 bp, a standard deviation of 15 bp, a minimum size of about 100 bp, and a maximum size of about 220 bp. The polymorphic sites of a particular target locus can occupy any position from start to end in the various fragments derived from this locus. Because the cfDNA fragments are short, the likelihood of the presence of two primer sites, including the likelihood of a fragment of length L with both forward and reverse primer sites, is the ratio of the amplicon length to the fragment length. Under ideal conditions, assays where the amplicon is 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, or 70 bp will successfully amplify from 72%, 69%, 66%, 63%, 59%, or 56% of the available template fragment molecules, respectively. In certain embodiments, cfDNA is amplified using primers that produce amplicons of 85 bp, 80 bp, 75 bp, or 70 bp and in certain preferred embodiments, a maximum amplicon length of 75 bp and having a melting temperature between 50°C and 65°C and in certain preferred embodiments, between 54°C - 60.5°C. The amplicon length is the distance between the 5' ends of the forward and reverse primer sites. Shorter amplicon lengths than typically used can enable more efficient measurement of the desired polymorphic loci by requiring only short sequence reads. In one embodiment, a substantial portion of the amplicon is less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp.

[0089] In some embodiments, the amplification is performed using direct multiplex PCR, sequence PCR, nested PCR, double nested PCR, hemi-nested PCR, full nested PCR, single-sided full nested PCR, single-sided nested PCR, semi-nested PCR, semi-nested PCR, triple semi-nested PCR, semi-nested PCR, single-sided semi-nested PCR, inverse semi-nested PCR, or single-sided PCR, as described in the following: U.S. Application Serial No. 13 / 683,604, filed November 21, 2012; U.S. Publication No. 2013 / 0123120; U.S. Application Serial 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 hereby incorporated by reference in their entireties. Optionally, any of these methods can be used for mini-PCR.

[0090] Optionally, the extension step of the PCR amplification can be limited from a temporal perspective to reduce amplification from fragments longer than 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, or 1,000 nucleotides. This can result in fragmentation or enrichment of shorter nucleic acids (such as cfDNA from cells undergoing apoptosis or necrosis) and improved test performance.

[0091] In some embodiments, multiplex PCR is used. In some embodiments, a method for amplifying target loci in a nucleic acid sample involves (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 100; 200; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci to produce a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to produce an amplification product containing the target amplicons. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the targeted loci are amplified. In various embodiments, less than 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, or 0.05% of the amplification products are primer dimers. In some embodiments, the primers are in solution (such as dissolved in the liquid phase rather than the solid phase). In some embodiments, the primers are in solution and are not immobilized on a solid support. In some embodiments, the primers are not part of a microarray. In some embodiments, the primers do not include molecular inversion probes (MIPs).

[0092] In some embodiments, two or more (such as 3 or 4) target amplicons (such as amplicons from the microPCR methods disclosed herein) are ligated together and then the ligation product is sequenced. Combining multiple amplicons into a single ligation product increases the efficiency of subsequent sequencing steps. In some embodiments, the target amplicons have a length less than 150, 100, 90, 75, or 50 base pairs before they are ligated. Selective enrichment and / or amplification can involve labeling each individual molecule with different tags, molecular barcodes, tags for amplification, and / or tags for sequencing. In some embodiments, the amplified products are analyzed by sequencing (such as by high-throughput sequencing) or by hybridization to an array (such as a SNP array, an ILLUMINA INFINIUM array, or an AFFYMETRIX gene chip). In some embodiments, nanopore sequencing is used, such as the nanopore sequencing technology developed by Genia (see, e.g., the World Wide Web URL geniachip.com / technology, which is hereby incorporated by reference in its entirety). In some embodiments, duplex sequencing is used (Schmitt et al., “Detection of ultra-rare mutations by next-generation sequencing,” Proc Natl Acad Sci USA. 109(36):14508-14513, 2012, which is hereby incorporated by reference in its entirety). This method greatly reduces errors by independently labeling and sequencing each of the two strands of the DNA duplex. Since the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors cause mutations in only one strand and can therefore be ignored as technical errors. In some embodiments, the method requires labeling both strands of the duplex DNA with a random but complementary double-stranded nucleotide sequence (called a duplex tag). The duplex tag sequence is incorporated into the sequencing adaptor by first introducing a single-stranded randomized nucleotide sequence into one adaptor strand and then extending the opposite strand with DNA polymerase to obtain a complementary, double-stranded tag. After the labeled adaptor is ligated to the sheared DNA, the individually labeled strands are PCR amplified from the asymmetric primer sites on the adaptor tails and subjected to paired-end sequencing. In some embodiments, a sample (such as a DNA sample) or a preparation of nucleic acids is divided into multiple portions, such as different wells (e.g., the wells of a WaferGen SmartChip). Dividing the sample or preparation into different portions (such as at least 5, 10, 20, 50, 75, 100, 150, 200, or 300 portions) can increase the sensitivity of the analysis because the percentage of molecules with mutations in some wells is higher compared to the overall sample.In some embodiments, each portion has less than 500, 400, 200, 100, 50, 20, 10, 5, 2, or 1 nucleic acid molecule. In some embodiments, the molecules in each portion are sequenced separately. In some embodiments, the same barcode (such as a random or non-human sequence) is added to all the molecules in the same portion (such as by amplification with a barcoded primer or by ligation of a barcode), and different barcodes are added to the molecules in different portions. The barcoded molecules can be combined and sequenced together. In some embodiments, the molecules are amplified prior to pooling and sequencing, such as by using nested PCR. In some embodiments, one forward and two reverse primers, or two forward and one reverse primer are used.

[0093] In some embodiments, less than 10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.005% of the DNA molecules in a sample (such as a sample of cfDNA) or a preparation of nucleic acids is measured to have a mutation (such as an SNV or CNV). In some embodiments, a mutation (such as an SNV or CNV) present in less than 1,000, 500, 100, 50, 20, 10, 5, 4, 3, or 2 original nucleic acid molecules (before amplification) in a sample (such as a sample of cfDNA from, for example, a blood sample, a perfusion fluid sample, or a lavage fluid sample) or a preparation of nucleic acids is measured to be present. In some embodiments, a mutation (such as an SNV or CNV) present in only 1 original nucleic acid molecule (before amplification) in a sample (such as a sample of cfDNA from, for example, a blood sample, a perfusion fluid sample, or a lavage fluid sample) or a preparation of nucleic acids is measured to be present.

[0094] For example, if the detection limit for a mutation (such as a single nucleotide variant (SNV)) is 0.1%, the mutation present at 0.01% can be measured by dividing the portion into multiple portions (such as 100 wells). Most of the wells do not have copies of the mutation. For the few wells that have the mutation, the mutation has a significantly higher read percentage. In one example, there are 20,000 initial copies of DNA from a target locus, and two of these copies include the relevant SNV. If the sample or preparation is divided into 100 wells, 98 wells have the SNV, and 2 wells have 0.5% SNV. The nucleic acids in each well can be barcoded, amplified, combined with the nucleic acids from other wells, and sequenced. The wells without the SNV can be used to measure the background amplification / sequencing error rate to evaluate whether the signal from the outlier wells is above the background noise level.

[0095] In some embodiments, an array is used to measure amplification products, such as an array having probes for one or more chromosomes of interest (e.g., chromosome 13, 18, 21, X, Y, or any combination thereof), particularly a microarray. For example, it should be understood that SNP detection microarrays can be used, such as, for example, Illumina (San Diego, Calif.) GoldenGate, DASL, Infinium, or CytoSNP-12 genotyping assays, or SNP detection microarray products from Affymetrix, such as the OncoScan microarray. In some embodiments, phased genetic data from one or both biological parents of an embryo or fetus is used to improve the accuracy of array data analysis of single cells.

[0096] In some embodiments involving sequencing, the read depth is the number of sequencing reads mapped to a given locus. The read depth can be normalized for the total number of reads. In some embodiments of the read depth of a sample or preparation, the read depth is the average read depth for the targeted locus. In some embodiments of the read depth of a locus, the read depth is the number of reads measured by a sequencer mapped to this locus. Generally, the greater the read depth of a locus, the more likely the ratio of alleles at the locus is to approach the ratio of alleles in the original sample or preparation of nucleic acid. The read depth can be expressed in a variety of different ways, including (but not limited to) percentages or proportions. Thus, for example, in a highly parallel DNA sequencer (such as an Illumina HISEQ) that generates, for example, 1 million clone sequences, 3,000 sequencings of a locus result in a read depth of 3,000 reads at that locus. The proportion of reads at this locus is 3,000 divided by 1 million total reads, or 0.3% of the total reads.

[0097] In some embodiments, allelic data is obtained, where the allelic data includes quantitative measurements indicative of the copy number of specific alleles of a polymorphic locus. In some embodiments, the allelic data includes quantitative measurements indicative of the copy number of each of the alleles observed at the polymorphic locus. Quantitative measurements can be made for all possible alleles of the polymorphic locus of interest. For example, any of the methods for assessing alleles of SNP or SNV loci discussed in the previous paragraph (such as, for example, microarrays, qPCR, RNA sequencing, DNA sequencing, such as high-throughput DNA sequencing) can be used to generate quantitative measurements of the copy number of specific alleles of a polymorphic locus. Such quantitative measurements are referred to herein as allelic frequency data or measured genetic allelic data. Methods that use allelic data are sometimes referred to as quantitative allelic methods; this is in contrast to quantitative methods that use only quantitative data from non-polymorphic loci or from polymorphic loci without considering allelic identity. When high-throughput sequencing is used to measure allelic data, the allelic data can include the number of reads mapped to each allele of the locus of interest.

[0098] In some embodiments, non-allelic data is obtained, where the non-allelic data includes quantitative measurements indicative of the copy number of a particular locus. The locus can be polymorphic or non-polymorphic. In some embodiments, when the locus is non-polymorphic, the non-allelic data does not contain information regarding the relative or absolute number of individual alleles that may be present at that locus. A method that uses only non-allelic data (i.e., quantitative data from non-polymorphic alleles, or from polymorphic loci without considering the allelic identity of each fragment) is called a quantitative method. Quantitative measurements of all possible alleles of a polymorphic locus of interest can be obtained, where a total of one value is associated with the measured number of all alleles at that locus. The non-allelic data for a polymorphic locus can be obtained by summing the quantitative alleles of each allele at that locus. When high-throughput sequencing is used to measure allelic data, the non-allelic data can include the number of reads mapped to the locus of interest. The sequencing measurements can indicate the relative and / or absolute number of each of the alleles present at that locus, and the non-allelic data includes the sum of the reads mapped to the locus regardless of allelic identity. In some embodiments, the same set of sequencing measurements can be used to generate both allelic data and non-allelic data. In some embodiments, allelic data is used as part of a method for assessing the copy number at a chromosome of interest, and the non-allelic data generated can be used as part of a different method for assessing the copy number at a chromosome of interest. In some embodiments, the two methods are statistically orthogonal and are combined to achieve a more precise assessment of the copy number at the chromosome of interest.

[0099] In some embodiments, obtaining genetic data includes (i) acquiring nucleic acid sequence information by laboratory techniques, such as by using an automated high-throughput sequencer, or (ii) obtaining information previously obtained by laboratory techniques, where the information is transmitted electronically, such as by a computer via the Internet or by electronic transfer from a sequencing device.

[0100] Additional exemplary sample preparation, amplification, and quantification methods are described in U.S. Patent Serial No. 13 / 683,604, filed November 21, 2012 (U.S. Publication No. 2013 / 0123120) and U.S. Serial No. 61 / 994,791, filed May 16, 2014, which are hereby incorporated by reference in their entireties. These methods can be used to analyze any of the samples or preparations disclosed herein.

[0101] Improved PCR amplification methods have also been developed that minimize or prevent interference caused by amplification of neighboring or adjacent target loci in the same reaction volume (such as part of a sample multiplex PCR reaction that simultaneously amplifies all target loci). These methods can be used to simultaneously amplify neighboring or adjacent target loci, which is faster and less costly compared to having to split neighboring target loci into separate reaction volumes so that they can be amplified individually to avoid interference.

[0102] In some embodiments, amplification of target loci is performed using a polymerase (e.g., a DNA polymerase or a reverse transcriptase) having low 5'→3' exonuclease and / or low strand displacement activity. In some embodiments, low levels of 5'→3' exonuclease reduce or prevent degradation of neighboring primers (e.g., unextended primers or primers to which one or more nucleotides have been added during primer extension). In some embodiments, low levels of strand displacement activity reduce or prevent displacement of neighboring primers (e.g., unextended primers or primers to which one or more nucleotides have been added during primer extension). In some embodiments, target loci that are adjacent to each other (e.g., no bases between the target loci) or neighboring (e.g., the loci are within 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base of each other) are amplified. In some embodiments, the 3' end of one locus is within 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base of the 5' end of the next downstream locus.

[0103] In some embodiments, at least 100, 200, 500, 750, 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified, such as by simultaneous amplification in one reaction volume. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the amplification products are target amplicons. In various embodiments, the amount of the amplification products that are target amplicons is between 50% and 99.5%, such as between 60% and 99%, 70% and 98%, 80% and 98%, 90% and 99.5%, or 95% and 99.5% and includes the end values. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the target loci are amplified (e.g., amplified at least 5, 10, 20, 30, 50 or 100 times compared to the amount before amplification), such as by simultaneous amplification in one reaction volume. In various embodiments, the amount of the target loci that are amplified (e.g., amplified at least 5, 10, 20, 30, 50 or 100 times compared to the amount before amplification) is between 50% and 99.5%, such as between 60% and 99%, 70% and 98%, 80% and 99%, 90% and 99.5%, 95% and 99.9%, or 98% and 99.99% (including the end values). In some embodiments, fewer non-target amplicons are produced, such as fewer amplicons formed by the forward primer from the first primer pair and the reverse primer from the second primer pair. Such undesirable non-target amplicons can be produced using prior amplification methods if, for example, the reverse primer from the first primer pair and / or the forward primer from the second primer pair are degraded and / or replaced.

[0104] In some embodiments, these methods allow for the use of longer extension times because, given the low 5'→3′ exonuclease and / or low strand displacement activity of the polymerase, the polymerase bound to the primer being extended is less likely to degrade and / or displace neighboring primers (such as the next downstream primer). In various embodiments, reaction conditions (such as extension time and temperature) are used such that the extension rate of the polymerase allows the number of nucleotides added to the primer being extended to be equal to or greater than 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 175% or 200% of the number of nucleotides between the 3' end of the primer binding site on the same strand and the 5' end of the next downstream primer binding site.

[0105] In some embodiments, DNA is used as a template and a DNA polymerase is used to generate DNA amplicons. In some embodiments, DNA is used as a template and an RNA polymerase is used to generate RNA amplicons. In some embodiments, RNA is used as a template and a reverse transcriptase is used to generate cDNA amplicons.

[0106] In some embodiments, under the same conditions, the low level of 5'→3′ exonuclease activity in a polymerase is less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the activity of the same amount of Thermus aquaticus polymerase (“Taq” polymerase, which is a commonly used DNA polymerase from a thermophilic bacterium, PDB 1BGX, EC 2.7.7.7, Murali et al., “Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix - coil dynamics of the enzyme,” Proc. Natl. Acad. Sci. USA 95:12562 - 12567, 1998, which is hereby incorporated by reference in its entirety). In some embodiments, under the same conditions, the low level of strand displacement activity in a polymerase is less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the activity of the same amount of Taq polymerase.

[0107] In some embodiments, the polymerase is a PUSHION DNA polymerase, such as PHUSION High-Fidelity DNA polymerase (M0530S, New England BioLabs, Inc.) or PHUSION Hot Start Flex DNA polymerase (M0535S, New England BioLabs, Inc.; Frey and Suppman BioChemica. 2:34-35, 1995; Chester and Marshak Analytical Biochemistry. 209:284-290, 1993, each of which is hereby incorporated by reference in its entirety). The PHUSION DNA polymerase is a Pyrococcus-like enzyme fused to a processivity-enhancing domain. The PHUSION DNA polymerase has 5'→3′ polymerase activity and 3'→5′ exonuclease activity and produces blunt-ended products. The PHUSION DNA polymerase lacks 5'→3′ exonuclease activity and strand displacement activity.

[0108] In some embodiments, the polymerase is a DNA polymerase, such as High-Fidelity DNA polymerase (M0491S, New England BioLabs, Inc.) or Hot Start High-Fidelity DNA polymerase (M0493S, New England BioLabs, Inc.). The High-Fidelity DNA polymerase is a high-fidelity, thermostable DNA polymerase with 3'→5′ exonuclease activity, which is fused to a processivity-enhancing Sso7d domain. The High-Fidelity DNA polymerase lacks 5'→3′ exonuclease activity and strand displacement activity.

[0109] In some embodiments, the polymerase is T4 DNA polymerase (M0203S, New England BioLabs, Inc.; Tabor and Struh. (1989). “DNA-Dependent DNA Polymerases,” see Ausebel et al. (eds.), Current Protocols in Molecular Biology. 3.5.10-3.5.12. New York: John Wiley & Sons, Inc., 1989; Sambrook et al. Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference in their entireties). T4 DNA polymerase catalyzes the synthesis of DNA in the 5'→3′ direction and requires the presence of a template and a primer. This enzyme has 3′→5′ exonuclease activity, which is significantly higher than the activity found in DNA polymerase I. T4 DNA polymerase lacks 5'→3′ exonuclease activity and strand displacement activity.

[0110] In some embodiments, the polymerase is Sulfolobus DNA polymerase IV (M0327S, New England BioLabs, Inc.; (Boudsocq et al. (2001). Nucleic Acids Res., 29:4607-4616, 2001; McDonald et al. (2006). Nucleic Acids Res., 34:1102-1111, 2006, each of which is hereby incorporated by reference in its entirety). Sulfolobus DNA polymerase IV is a thermostable Y-family lesion bypass DNA polymerase that efficiently synthesizes DNA across a variety of DNA template lesions (McDonald, J.P. et al. (2006). Nucleic Acids Res., 34, 1102-1111, which is hereby incorporated by reference in its entirety). Sulfolobus DNA polymerase IV lacks 5′→3′ exonuclease activity and strand displacement activity.

[0111] In some embodiments, if a primer binds to a region with an SNP, the primer may bind and amplify different alleles with different efficiencies or may only bind and amplify one allele. For a heterozygous subject, one allele may not be amplified by the primer. In some embodiments, primers are designed for each allele. For example, if there are two alleles (e.g., a biallelic SNP), two primers can be used to bind to the same position of the target locus (e.g., a forward primer for binding the "A" allele and a forward primer for binding the "B" allele). Methods such as, but not limited to, the dbSNP database can be used to evaluate the positions of known SNPs (such as SNP hotspots with a high heterozygosity rate).

[0112] In some embodiments, the amplicons are similar in size. In some embodiments, the length of the target amplicon ranges from less than 100, 75, 50, 25, 15, 10, or 5 nucleotides. In some embodiments (such as the amplification of a target locus in fragmented nucleic acids), the length of the target amplicon is between 50 and 100 nucleotides, such as between 60 and 80 nucleotides or between 60 and 75 nucleotides (including the end values). In some embodiments (such as the amplification of multiple target loci in an entire exon or gene), the length of the target amplicon is between 100 and 500 nucleotides, such as between 150 and 450 nucleotides, between 200 and 400 nucleotides, between 200 and 300 nucleotides, or between 300 and 400 nucleotides and including the end values).

[0113] In some embodiments, multiple target loci are simultaneously amplified using a primer pair that includes forward and reverse primers for each target locus to be amplified in the reaction volume. In some embodiments, each target locus is subjected to one round of PCR with a single primer and then each target locus is subjected to a second round of PCR with a primer pair. For example, each target locus can be subjected to one round of PCR with a single primer such that all primers bind to the same strand (such as using forward primers for each target locus). This allows PCR to amplify in a linear manner and reduces or eliminates amplification bias caused by sequence or length differences between amplicons. In some embodiments, forward and reverse primers are then used for each target locus to amplify the amplicon.

[0114] As needed, primers with a reduced likelihood of forming primer dimers can be used for multiplex PCR. In particular, highly multiplex PCR typically results in a very high proportion of product nucleic acids generated by non-productive side reactions such as primer dimer formation. In one embodiment, specific primers that are most likely to cause non-productive side reactions can be removed from the primer library, resulting in a primer library that will produce a greater proportion of amplified nucleic acids that map to the genome. The step of removing problematic primers (i.e., primers that are particularly likely to form dimers) has unexpectedly achieved extremely high levels of PCR multiplexing for subsequent analysis by sequencing.

[0115] There are a variety of ways to select primers for a library such that the amount of non-mapping primer dimers or other primer failure products is minimized. Empirical data indicates that a small number of 'bad' primers cause a large number of non-mapping primer dimer side reactions. Removing these 'bad' primers can increase the percentage of sequence reads that map to the target locus. One way to identify 'bad' primers is to look at the sequencing data of the nucleic acids amplified by targeted amplification; primers dimers that occur most frequently can be removed, resulting in a primer library that is less likely to produce byproduct nucleic acids that do not map to the genome. There are also programs that can calculate the binding energies of various primer combinations, and removing those primer combinations with the highest binding energies will also result in a primer library that is significantly less likely to produce byproduct nucleic acids that do not map to the genome.

[0116] In some embodiments for selecting primers, an initial candidate primer library is created by designing one or more primers or primer pairs to be candidate target loci. A set of candidate target loci (such as SNPs) can be selected based on information about desired parameters of the target loci, such information being, for example, the frequency of the SNP within the target population or the heterozygosity rate of the SNP. In one embodiment, PCR primers can be designed using the Primer3 program (World Wide Web URL primer3.sourceforge.net; libprimer3 version 2.2.3, which is hereby incorporated by reference in its entirety). Optionally, primers can be designed to anneal within a specific annealing temperature range, have a specific range of GC content, have a specific size range, produce target amplicons within a specific size range, and / or have other parameter characteristics. Starting with multiple primers or primer pairs for each candidate target locus increases the likelihood that the primers or primer pairs will remain in the library for most or all of the target loci. In one embodiment, the selection criteria may require that at least one primer pair for each target locus remain in the library. In this way, most or all of the target loci will be amplified when using the final primer library. This is what is needed for applications such as screening for deletions or duplications at a large number of positions in the genome, or screening for a large number of sequences (such as polymorphisms or other mutations) associated with a disease or increased disease risk. If primer pairs from the library will produce target amplicons that overlap with target amplicons produced by another primer pair, one of the primer pairs can be removed from the library to prevent interference.

[0117] In some embodiments, the “undesirability score” (a higher score indicates less desirability) of most or all possible combinations of two primers from a candidate primer library is calculated (such as on a computer). In various embodiments, the undesirability scores of at least 80%, 90%, 95%, 98%, 99%, or 99.5% of the possible candidate primer combinations in the library are calculated. Each undesirability score is at least partially based on the likelihood of forming a dimer between the two candidate primers. Optionally, the undesirability score may also be based on one or more other parameters selected from the group consisting of: the heterozygosity rate of the target locus, the disease prevalence associated with the sequence at the target locus (e.g., polymorphism), the disease penetrance associated with the sequence at the target locus (e.g., polymorphism), the specificity of the candidate primer for the target locus, the size of the candidate primer, the melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, the size of the target amplicon, and the distance from the center of the recombination hot spot. In some embodiments, the specificity of the candidate primer for the target locus includes the likelihood of the candidate primer mispriming due to binding and amplifying loci other than the target locus it is designed to amplify. In some embodiments, one or more or all of the candidate primers that misprime are removed from the library. In some embodiments, in order to increase the number of candidate primers selected, candidate primers that may misprime are not removed from the library. If multiple factors are considered, the undesirability score can be calculated based on a weighted average of various parameters. The parameters can be assigned different weights based on the importance of the parameter for the specific application in which the primers will be used. In some embodiments, the primers with the highest undesirability scores are removed from the library. If the removed primer is a member of a primer pair that hybridizes to a target locus, the other member of the primer pair can be removed from the library. The process of removing primers can be repeated as needed. In some embodiments, the selection method is performed until the undesirability scores of the remaining candidate primer combinations in the library are all equal to or below a minimum threshold. In some embodiments, the selection method is performed until the number of remaining candidate primers in the library is reduced to the desired number.

[0118] In various embodiments, after calculating the undesirability scores, candidate primers that are part of the maximum number of combinations of two candidate primers and have an undesirability score higher than a first minimum threshold are removed from the library. This step ignores interactions that are equal to or below the first minimum threshold because these interactions are less significant. If the removed primer is a member of a primer pair that hybridizes to a target locus, the other member of the primer pair can be removed from the library. The process of removing primers can be repeated as needed. In some embodiments, the selection method is carried out until the undesirability scores of all the remaining candidate primer combinations in the library are equal to or below the first minimum threshold. If the number of remaining candidate primers in the library is higher than the desired number, the number of primers can be reduced by lowering the first minimum threshold to a lower second minimum threshold and repeating the process of removing primers. If the number of remaining candidate primers in the library is lower than the desired number, the method can be continued by increasing the first minimum threshold to a higher second minimum threshold and repeating the process of removing primers using the original candidate primer library, thereby allowing more candidate primers to remain in the library. In some embodiments, the selection method is carried out until the undesirability scores of all the remaining candidate primer combinations in the library are equal to or below the second minimum threshold, or until the number of remaining candidate primers in the library is reduced to the desired number.

[0119] Optionally, primer pairs that produce target amplicons that overlap with target amplicons produced by another primer pair can be assigned to separate amplification reactions. For applications that require analysis of all candidate target loci (as opposed to omitting candidate target loci from the analysis due to overlapping target amplicons), multiple PCR amplification reactions may be required.

[0120] These selection methods minimize the number of candidate primers that must be removed from the library, achieving the desired reduction in primer dimers. By removing a smaller number of candidate primers from the library, more (or all) of the target loci can be amplified using the resulting primer library.

[0121] Multiplexing a large number of primers places a significant number of restrictions on the assays that can be included. Assays that interact inadvertently can produce false amplification products. The size limitations of miniPCR can impose further restrictions. In one embodiment, it is possible to start with a very large number of potential SNP targets (between about 500 and greater than 1 million) and attempt to design primers that amplify each SNP. When primers can be designed, it is possible to attempt to identify primer pairs that are likely to form false primer duplexes by evaluating the likelihood of forming false primer duplexes between all possible primer pairs using the published thermodynamic parameters for duplex formation. Primer interactions can be ranked by a scoring function associated with the interaction and the primers with the worst interaction scores are eliminated until the desired number of primers is met. In cases where SNPs are most likely to be heterozygous, it is also possible to rank the assay list and select the assays with the highest heterozygous compatibility. Experiments have verified that primers with high interaction scores are most likely to form primer dimers. At high multiplexing levels, it is not possible to eliminate all false interactions, but it is necessary to remove the primers or primer pairs with the highest interaction scores in computer simulations because they will dominate the entire reaction and greatly limit the amplification of the intended targets. This procedure has been carried out to create multiplex primer sets with up to and in some cases, more than 10,000 primers. Due to this procedure, the improvements are substantial, achieving more than 80%, more than 90%, more than 95%, more than 98% and even more than 99% amplification of the target product compared to 10% from reactions where the worst primers were not removed, as measured by sequencing of all PCR products. When combined with the partial semi-nested method as previously described, more than 90% and even more than 95% of the amplicons can be mapped to the targeted sequences.

[0122] It should be noted that there are other methods for evaluating which PCR probes are likely to form dimers. In an embodiment, analyzing a pool of nucleic acids amplified using a set of non-optimized primers may be sufficient to identify problematic primers. For example, sequencing can be used for the analysis, and the dimers present in the largest numbers are identified as most likely to form dimers and can be removed. In one embodiment, the primer design method can be used in combination with the miniPCR method described herein.

[0123] Using a tag on a primer can reduce the amplification and sequencing of primer dimer products. In some embodiments, the primer contains an internal region that forms a loop structure with the tag. In certain embodiments, the primer includes a 5′ region that is specific for a target locus, an internal region that is not specific for the target locus and forms a loop structure, and a 3′ region that is specific for the target locus. In some embodiments, the loop region can be between two binding regions, where the two binding regions are designed to bind to adjacent or neighboring regions of the template nucleic acid. In various embodiments, the length of the 3′ region is at least 7 nucleotides. In some embodiments, the length of the 3′ region is between 7 and 20 nucleotides, such as between 7 and 15 nucleotides or between 7 and 10 nucleotides (including the end values). In various embodiments, the primer includes a 5′ region that is not specific for the target locus (such as a tag or a universal primer binding site), followed by a region that is specific for the target locus, an internal region that is not specific for the target locus and forms a loop structure, and a 3′ region that is specific for the target locus. The tag-primers can be used to shorten the required target-specific sequences to less than 20, less than 15, less than 12, and even less than 10 base pairs. This can be the case in primer design when fragmenting the target sequence within the primer binding site or, alternatively, when the target sequence can be serendipitously designed into the primer design. Advantages of this method include: the method increases the number of assays that can be designed for a given maximum amplicon length, and the method shortens the "non-informative" sequencing of the primer sequence. The method can also be used in combination with internal labels.

[0124] In embodiments, the relative amount of non-productive products in multiplexed PCR amplification can be reduced by raising the annealing temperature. In the case of an amplification library containing tags identical to the target-specific primers, the annealing temperature can be increased relative to genomic DNA because the tags will result in primer binding. In some embodiments, a reduced primer concentration is used, optionally in combination with a longer annealing time. In some embodiments, the annealing time can be more than 3 minutes, more than 5 minutes, more than 8 minutes, more than 10 minutes, more than 15 minutes, more than 20 minutes, more than 30 minutes, more than 60 minutes, more than 120 minutes, more than 240 minutes, more than 480 minutes and even more than 960 minutes. In certain illustrative embodiments, a longer annealing time and a reduced primer concentration are used. In various embodiments, a time greater than normal extension is used, such as greater than 3 minutes, 5 minutes, 8 minutes, 10 minutes or 15 minutes. In some embodiments, the primer concentration is as low as 50 nM, 20 nM, 10 nM, 5 nM, 1 nM and below 1 nM. This unexpectedly results in a robust performance of highly multiplexed reactions, such as 1,000-plex reactions, 2,000-plex reactions, 5,000-plex reactions, 10,000-plex reactions, 20,000-plex reactions, 50,000-plex reactions and even 100,000-plex reactions. In one embodiment, the amplification uses one, two, three, four or five cycles run with a long annealing time, followed by PCR cycles run with a more common annealing time and labeled primers.

[0125] To select target locations, one can start with a pool of candidate primer pairs design and create a thermodynamic model of potential adverse interactions between the primer pairs, and then use the model to eliminate designs that are incompatible with other designs in the pool.

[0126] In an embodiment, the system or method features a method of reducing the number of target loci (such as loci that may contain polymorphisms or mutations associated with a disease or disorder or an increased risk of transplant rejection) and / or increasing the measured disease burden (e.g., increasing the number of measured polymorphisms or mutations). In some embodiments, the method includes ranking (such as ranking from highest to lowest) the loci by the frequency or recurrence of polymorphisms or mutations (such as single nucleotide changes, insertions or deletions or any other changes described herein) in each locus in a subject having a disease or disorder. In some embodiments, PCR primers are designed to target some or all of the loci. During selection of the PCR primers of the primer library, primers targeting loci with higher frequency or recurrence (higher ranked loci) are favored compared to loci with lower frequency or recurrence (lower ranked loci). In some embodiments, this parameter is included as one of the parameters in the calculation of the undesirability score described herein. Optionally, primers that are incompatible with other designs in the library (such as primers targeting high ranked loci) may be included in different PCR libraries / pools. In some embodiments, multiple libraries / pools (such as 2, 3, 4, 5 or more) are used in separate PCR reactions to achieve amplification of all (or most) of the loci represented by all the libraries / pools. In some embodiments, this method is continued until sufficient primers are included in one or more of the libraries / pools such that the combined primers can achieve the desired disease burden for capturing the disease or disorder (e.g., such as by measuring at least 80%, 85%, 90%, 95% or 99% of the disease burden).

[0127] In some embodiments, the systems or methods described herein use a primer library, such as primers selected from a candidate primer library using any of the methods described herein. In some embodiments, the library includes primers that simultaneously hybridize (or are capable of simultaneously hybridizing) or simultaneously amplify (or are capable of simultaneously amplifying) at least 100; 200; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci in a single reaction volume. In various embodiments, the library includes primers that simultaneously amplify (or are capable of simultaneously amplifying) 100 to 500; 500 to 1,000; 1,000 to 2,000; 2,000 to 5,000; 5,000 to 7,500; 7,500 to 10,000; 10,000 to 20,000; 20,000 to 25,000; 25,000 to 30,000; 30,000 to 40,000; 40,000 to 50,000; 50,000 to 75,000; or 75,000 to 100,000 different target loci (including the end values) in a single reaction volume. In various embodiments, the library includes primers that simultaneously amplify (or are capable of simultaneously amplifying) between 1,000 and 100,000 different target loci in a single reaction volume, such as 1,000 to 50,000; 1,000 to 30,000; 1,000 to 20,000; 1,000 to 10,000; 2,000 to 30,000; 2,000 to 20,000; 2,000 to 10,000; 5,000 to 30,000; 5,000 to 20,000; or 5,000 to 10,000 different target loci (including the end values). In some embodiments, the library includes primers that simultaneously amplify (or are capable of simultaneously amplifying) target loci in a single reaction volume such that less than 60%, 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 various embodiments, the amount of amplification products that are primer dimers is between 0.5% and 60%, such as between 0.1% and 40%, 0.1% and 20%, 0.25% and 20%, 0.25% and 10%, 0.5% and 20%, 0.5% and 10%, 1% and 20%, or 1% and 10% and includes the end values. In some embodiments, the primers simultaneously amplify (or are capable of simultaneously amplifying) target loci in a single reaction volume such that at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the amplification products are target amplicons.In various embodiments, the amount of the amplification product that is the target amplicon is between 50% and 99.5%, such as between 60% and 99%, 70% and 98%, 80% and 98%, 90% and 99.5%, or 95% and 99.5% and including the end values. In some embodiments, the primers co-amplify (or are capable of co-amplifying) the target locus in a reaction volume such that at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the target locus is amplified (e.g., amplified at least 5, 10, 20, 30, 50, or 100-fold compared to the amount prior to amplification). In various embodiments, the amount of the target locus that is amplified (e.g., amplified at least 5, 10, 20, 30, 50, or 100-fold compared to the amount prior to amplification) is between 50% and 99.5%, such as between 60% and 99%, 70% and 98%, 80% and 99%, 90% and 99.5%, 95% and 99.9%, or 98% and 99.99% (including the end values). In some embodiments, the library of primers includes at least 100; 200; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 primer pairs, where each pair of primers includes a forward test primer and a reverse test primer, and where each pair of test primers hybridizes to the target locus. In some embodiments, the library of primers includes at least 100; 200; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 individual primers, each of which hybridizes to a different target locus, where the individual primers are not part of a primer pair.

[0128] In various embodiments, the concentration of each primer is less than 100 nM, 75 nM, 50 nM, 25 nM, 20 nM, 10 nM, 5 nM, 2 nM, or 1 nM, or less than 500 μM, 100 μM, 10 μM, or 1 μM. In various embodiments, the concentration of each primer is between 1 μM and 100 nM, such as between 1 μM and 1 nM, 1 nM and 75 nM, 2 nM and 50 nM, or 5 nM and 50 nM and including the end values. In various embodiments, the GC content of the primer is between 30% and 80%, such as between 40% and 70% or 50% and 60% and including the end values. In some embodiments, the GC content of the primer ranges from less than 30%, 20%, 10%, or 5%. In some embodiments, the GC content of the primer ranges between 5% and 30%, such as between 5% and 20% or 5% and 10% and including the end values. In some embodiments, the melting temperature (Tm) of the test primer is between 40 °C and 80 °C, such as between 50 °C and 70 °C, 55 °C and 65 °C, or 57 °C and 60.5 °C (including the end values). In some embodiments, the Primer3 program (libprimer3 version 2.2.3) is used with built-in SantaLucia parameters (World Wide Web URL primer3.sourceforge.net) to calculate the Tm. In some embodiments, the melting temperature of the primer ranges from less than 15 °C, 10 °C, 5 °C, 3 °C, or 1 °C. In some embodiments, the melting temperature of the primer ranges between 1 °C and 15 °C, such as between 1 °C and 10 °C, 1 °C and 5 °C, or 1 °C and 3 °C (including the end values). In some embodiments, the length of the primer is between 15 and 100 nucleotides, such as between 15 and 75 nucleotides, 15 and 40 nucleotides, 17 and 35 nucleotides, 18 and 30 nucleotides, or 20 and 65 nucleotides and including the end values. In some embodiments, the length of the primer ranges from less than 50, 40, 30, 20, 10, or 5 nucleotides. In some embodiments, the length of the primer ranges between 5 and 50 nucleotides, such as between 5 and 40 nucleotides, 5 and 20 nucleotides, or 5 and 10 nucleotides and including the end values. In some embodiments, the length of the target amplicon is between 50 and 100 nucleotides, such as between 60 and 80 nucleotides or 60 to 75 nucleotides and including the end values. In some embodiments, the length of the target amplicon ranges from less than 50, 25, 15, 10, or 5 nucleotides. In some embodiments, the length of the target amplicon ranges between 5 and 50 nucleotides, such as between 5 and 25 nucleotides, 5 and 15 nucleotides, or 5 and 10 nucleotides and including the end values. In some embodiments, the library does not include a microarray. In some embodiments, the library includes a microarray.

[0129] In some embodiments, in addition to naturally occurring phosphodiester bonds, some (such as at least 80%, 90%, or 95%) or all of the linkers or primers include one or more bonds between adjacent nucleotides. Examples of such bonds include phosphoramide, phosphorothioate, and dithiophosphate bonds. In some embodiments, some (such as at least 80%, 90%, or 95%) or all of the linkers or primers include a phosphorothioate (such as a monothiophosphate) between the last 3' nucleotide and the penultimate 3' nucleotide. In some embodiments, some (such as at least 80%, 90%, or 95%) or all of the linkers or primers include phosphorothioates (such as monothiophosphates) between the last 2, 3, 4, or 5 nucleotides at the 3' end. In some embodiments, some (such as at least 80%, 90%, or 95%) or all of the linkers or primers include phosphorothioates (such as monothiophosphates) between at least 1, 2, 3, 4, or 5 of the last 10 nucleotides at the 3' end. In some embodiments, such primers are less likely to cleave or degrade. In some embodiments, the primers do not contain enzyme cleavage sites (such as protease cleavage sites).

[0130] Additional exemplary multiplex PCR methods and libraries are described in the following: U.S. Application Serial No. 13 / 683,604, filed November 21, 2012 (U.S. Publication No. 2013 / 0123120) and U.S. Serial No. 61 / 994,791, filed May 16, 2014, each of which is hereby incorporated by reference in its entirety). These methods and libraries can be used to analyze any of the samples or preparations disclosed herein and in any method of the present disclosure.

[0131] Example #1

[0132] Figures 5A to 9B Depicts the results of a study conducted on perfusion fluid samples collected from donated kidneys. A group of seven donor kidneys, where four people each donated one kidney and three people each donated two kidneys. The kidney samples are listed in Table 1. Four 10 mL aliquots were taken from 10 perfusions of these kidneys. Each aliquot was centrifuged to separate the cell pellet from the rest of the perfusion fluid. Cell DNA was extracted from the cell pellet using a DNA Micro Kit, and cfDNA was extracted from the perfusion fluid using the "NICE Chemistry" protocol. The extracted DNA was quantified by an Invitrogen fluorometer (using the dsDNA BR Assay Kit), and the fragment size was evaluated by an Agilent 2100 system (using the High Sensitivity DNA Assay and the DNA-1000 Assay).

[0133] Table 1: Perfusion times and temperatures of the kidney samples used in the study.

[0134] Subject ID Kidney Perfusion time (min) Temperature °C AHFK479 - - – AHFL447 Right - 1.7 AHFL447 Left - 1.2 AHFK342 Right 744 2.0 AHF2108 Left 717 1.3 AHF3305 Left 1265 2.1 AHF3305 Right 1658 2.7 AHGC414 Right 1020 1.5 AHGC414 Left 1145 1.5 AHGF262 Left 660 2.7

[0135] Figure 5A and Figure 5B respectively show the electrophoretograms of the cell-free and cellular DNA fractions from each kidney perfusion fluid sample. The horizontal axis represents the fragment size in base pairs (bp), and the vertical axis represents the relative level of each fragment size based on fluorescence units (FU). In Figure 5A , the subscript (LM) shows a spike at 15 bp, and the superscript (UM) shows a spike at 1500. In Figure 5B , LM shows a spike at 35 bp, and UM shows a spike at 10,380 bp. LM and UM are internal markers added during processing to ensure the proper sizing of the samples and do not represent the levels of sample DNA.

[0136] As Figure 5A can be seen, cfDNA exists in the perfusion fluid samples as fragments of different sizes, represented by several small peaks around 200 bp and 400 bp and higher molecular weights. This distribution of smaller cfDNA fragments (e.g., less than about 200 bp) can indicate that DNA is fragmented around nucleosomes and released from cells during apoptosis, as these peaks have the same height and are present at equal intervals throughout the spectrum corresponding to the nucleosome size. In Figure 5B , cellular DNA exists almost entirely as high molecular weight fragments (e.g., greater than 200 bp).

[0137] Figure 6A , Figure 6B and Figure 6C show the electrophoretograms of specific samples with different size profiles. In Figure 6A , the cfDNA size profile of a sample that was evaluated as having the highest DNA concentration among the full set of samples is shown. This specific sample has only detectable levels of high molecular weight fragments, while any low molecular weight (nucleosome) fragments are at undetectable levels. The corresponding cellular DNA profile is shown in Figure 6B , where only detectable levels of high molecular weight fragments are present as well. Figure 6C shows the cfDNA electrophoretogram of a specific sample AHGF262_L, which clearly has a peak at the 166 bp fragment, indicating the presence of a large amount of nucleosomal DNA.

[0138] The presence of nucleosome peaks in the samples can be used to evaluate the possible source of cfDNA, which can be related to the health status of the donor organ / tissue. For example, Figure 6A and Figure 6BSamples therein may be labeled as mechanical organ / tissue damage due to surgery or immune cell release, but there is a lack of nucleosome fragmentation in cfDNA (e.g., a characteristic of cell necrosis), while Figure 6C Samples with fragment sizes of approximately 160 - 170 bp in

[0139] may be due to programmed cell death caused by apoptosis, which may persist after transplantation, while the DNA in other representative samples may be the result of necrosis. Figure 7 In

[0140] a graph is plotted of the cellular DNA yield (in μg / L) versus the cfDNA yield (in μg / L) for each sample. The amount of cellular DNA in each sample is on average only about 1.3% of the amount of cfDNA; however, there is a linear correlation between the relative yields of each portion of DNA. Thus, it may be possible to use the quantity of cellular DNA or cfDNA to estimate the quantity of the other. The slope of the linear correlation is 0.013, the y-intercept is 3.61, and the R-squared (coefficient of determination) value is 0.61. Figure 8 In Figure 6A and Figure 6B the percentage of cfDNA yield relative to kidney weight for each sample is graphed. The average kidney weight is reported to be 130 g. The two raised bars are samples taken from the two kidneys of donor AHF3305, whose electrophoretograms are shown in

[0141] In Figure 9A and Figure 9B graphs are plotted of the yields of cfDNA and cellular DNA (in μg / L) versus the perfusion time (the length of time each kidney was perfused until the perfusion fluid sample was collected) (in hours), respectively. In Figure 9A there is a strong linear correlation between the cfDNA yield and the perfusion time. The slope of the linear correlation is 124, the y-intercept is 1470, and the R-squared value is 0.9. In Figure 9B the slope of the linear correlation between the cellular DNA yield and the perfusion time is 1.5, the y-intercept is 13.1, and the R-squared value is 0.48. It is noteworthy that the sample with the longest perfusion time is the same sample that shows the Figure 8 highest yield of cfDNA in

[0142] The foregoing is only for illustrative purposes of the principles of the present disclosure, and the device may be practiced by other embodiments in addition to the described embodiments, which are presented for illustrative purposes only and not for purposes of limitation. It should be understood that the device disclosed herein, while shown for the mechanical perfusion of donor organs or tissues, may also be applied to the perfusion of other biological entities.

[0143] Those skilled in the art will think of variations and modifications after reading this disclosure. The disclosed features can be implemented in any combination and sub - combination (including multiple dependent combinations and sub - combinations) with one or more other features described herein. The various features (including any components thereof) described or illustrated above can be combined or integrated into other systems. In addition, certain features may be omitted or not implemented.

[0144] The described systems and methods can be implemented by a doctor or automatically on a mechanical perfusion system and / or a DNA analysis platform. The perfusion system and / or the DNA analysis platform can include a data - processing device. The systems and methods described herein can be implemented remotely on a separate data - processing device. The separate data - processing device can be directly or indirectly connected to the system / platform through a cloud application. The system / platform can communicate with the separate data - processing device in real - time (or near real - time).

[0145] In general, embodiments of the subject matter and functional operations described in this specification can be implemented in digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in a combination of one or more of the foregoing components. Embodiments of the subject matter described in this specification can be implemented as one or more computer program products, i.e., one or more modules of computer program instructions encoded on a computer - readable medium for execution by, or to control the operation of, a data - processing apparatus. The computer - readable medium can be a machine - readable storage device, a machine - readable storage substrate, a memory device, a composition of matter affecting a machine - readable propagated signal, or a combination of one or more of them. The term “data - processing apparatus” encompasses all apparatus, devices, and machines for processing data, including, by way of example, a programmable processor, a computer, or multiple processors or computers. The apparatus can include, in addition to hardware, code that creates an execution environment for the computer programs being discussed, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, or a combination of one or more of them. A propagated signal is an artificially generated signal, such as an electrical, optical, or electromagnetic signal generated by a machine for the purpose of encoding information for transmission to a suitable receiver device.

[0146] A computer program (also referred to as a program, software, software application, script, or code) can be written in any form of programming language (including compiled or interpreted languages), and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program can correspond to a file in a file system. The program can be stored as part of a file that holds other programs or data (such as one or more scripts stored in a markup language file), stored in a single file dedicated to the program being discussed, or stored in multiple coordinated files (such as files that store one or more modules, subroutines, or portions of code). A computer program can be deployed to execute on one computer or on multiple computers located at one site or distributed across multiple sites and interconnected by a communication network.

[0147] The processes and logical flows described in this specification can be performed by one or more programmable processors that execute one or more computer programs to perform functions by operating on input data and generating output. The processes and logical flows can also be performed by special-purpose logic circuitry (such as a field-programmable gate array (FPGA) or an application-specific integrated circuit (ASIC)), and the apparatus can also be implemented as special-purpose logic circuitry.

[0148] Processors suitable for executing a computer program include, by way of example, both general and special-purpose microprocessors, as well as any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. The basic elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include one or more mass storage devices for storing data (such as magnetic disks, magneto-optical disks, or optical disks) or be operatively coupled to receive data from or transfer data to a mass storage device or both. However, a computer need not have such devices.

[0149] Those skilled in the art can identify instances of changes, substitutions, and alterations, and can make changes, substitutions, and alterations without departing from the scope of the information disclosed herein. All references cited herein are incorporated by reference in their entirety and become a part of this application.

Claims

1. A method for preparing a sample of non-naturally occurring nucleic acids from a perfusion fluid or a rinsing fluid, the nucleic acid sample being useful for assessing the transplantation outcome of a donor organ or a donor tissue, the method comprising: obtaining a sample of the perfusion fluid or the rinsing fluid from a donor organ or a donor tissue that has been perfused with the perfusion fluid or prepared with the rinsing fluid, the perfusion fluid or the rinsing fluid comprising nucleic acids; isolating all nucleic acids from the sample; and preparing the isolated nucleic acids for determination to measure at least one of the amount of nucleic acids in the perfusion fluid or the rinsing fluid, the molecular weight of the nucleic acids in the perfusion fluid or the rinsing fluid, or the fragment size distribution of the nucleic acids in the perfusion fluid or the rinsing fluid.

2. The method according to claim 1, wherein the amount of the nucleic acids is the total amount of DNA in the perfusion fluid or the rinsing fluid.

3. The method according to claim 1, wherein the amount of the nucleic acids is the amount of high molecular weight DNA in the perfusion fluid or the rinsing fluid.

4. The method according to claim 3, wherein the high molecular weight DNA comprises DNA fragments having more than 200 base pairs.

5. The method according to claim 1, wherein the amount of the nucleic acids is the amount of low molecular weight DNA in the perfusion fluid or the rinsing fluid.

6. The method according to claim 3, wherein the low molecular weight DNA comprises DNA fragments having less than 200 base pairs.

7. The method according to claim 1, wherein the molecular weight of the DNA is the average molecular weight of the DNA in the perfusion fluid or the rinsing fluid.

8. The method according to any one of claims 1 to 7, wherein the isolated nucleic acids are cell-free DNA.

9. The method according to claim 8, further comprising: if the cell-free DNA has a molecular weight consistent with nucleosomal DNA, assessing that the cell-free DNA is derived from apoptosis.

10. The method according to any one of claims 1 to 7, wherein the isolated nucleic acids are cellular DNA.

11. The method according to any one of claims 1 to 7, wherein the isolated nucleic acids are RNA.

12. The method according to claim 11, wherein the method further comprises: assessing a gene expression profile corresponding to the isolated RNA to evaluate the cellular origin of the RNA.

13. The method according to claim 12, wherein when the gene expression profile is related to immune function, the cellular origin of the RNA is evaluated as an immune cell.

14. The method according to any one of claims 11 to 13, wherein isolating the RNA comprises lysing vesicles containing the RNA.

15. The method according to any one of claims 1 to 14, further comprising: normalizing the amount of the nucleic acids relative to the perfusion time.

16. The method according to any one of claims 1 to 15, further comprising: normalizing the amount of the nucleic acids relative to at least one of the size, weight, volume or surface area of the donor organ or the donor tissue.

17. The method according to any one of claims 1 to 16, further comprising: Normalize the amount of DNA relative to the perfusion volume of the perfusion fluid or the flushing volume of the flushing fluid.

18. The method according to any one of claims 1 to 17, further comprising: Normalize the amount of the nucleic acid relative to the temperature of the perfusion fluid or the flushing fluid.

19. The method according to any one of claims 1 to 18, further comprising: Evaluate the fragmentation pattern of the nucleic acid.

20. The method according to claim 19, further comprising: Based on the fragmentation pattern, classify the isolated nucleic acid into randomly degraded nucleic acid from lysed cells in the perfusion fluid or the flushing fluid and apoptotic nucleic acid from cells in the donor organ or donor tissue.

21. The method according to any one of claims 1 to 20, further comprising: Obtain one or more additional samples of the perfusion fluid or the flushing fluid collected at a different time point from the sample; Quantify the amount of nucleic acid in the perfusion fluid or the flushing fluid of the one or more additional samples; And Track the amount of the nucleic acid in the perfusion fluid or the flushing fluid over time based on the sample and the one or more additional samples.

22. The method according to any one of claims 1 to 21, wherein the measurement comprises at least one of electrophoresis, mass spectrometry, fluorometry, qPCR or droplet PCR.

23. The method according to any one of claims 1 to 22, which comprises assaying the prepared isolated nucleic acid for measuring at least one of the amount of nucleic acid in the perfusion fluid or the flushing fluid, the molecular weight of the nucleic acid in the perfusion fluid or the flushing fluid, or the fragment size distribution of the nucleic acid in the perfusion fluid or the flushing fluid.

24. A method for assessing the outcome of a transplantation, the method comprising: Prepare a nucleic acid preparation according to the method of any one of claims 1 to 23; And Assess the outcome of the transplantation of the donor organ or donor tissue based on the amount of the nucleic acid.

25. The method according to claim 24, wherein the nucleic acid is cellular DNA from donor-derived cells, and wherein the predicted outcome is based on the predicted favorable or unfavorable prognosis of the cellular DNA, the cellular DNA indicating an immune response.

26. The method according to claim 24, wherein the nucleic acid is cell-free DNA, and wherein the predicted outcome is based on the predicted favorable or unfavorable prognosis of the cell-free DNA, the cell-free DNA indicating damage to the donor organ or donor tissue.

27. The method according to claim 24, wherein the predicted outcome is a predicted unfavorable prognosis, the predicted unfavorable prognosis being for a decision maker not to use the organ for transplantation.

28. The method according to any one of claims 24 to 27, wherein the predicted outcome is predicted at least in part based on a risk determination prediction determined by an algorithm that uses the amount of the nucleic acid as an algorithm input.

29. The method according to any one of claims 24 to 28, wherein the predicted outcome is predicted by a quality assessment of the donor organ or donor tissue based on the amount of the nucleic acid.

30. The method according to claim 29, wherein the quality assessment indicates at least one of the following characteristics associated with the transplantation outcome: the presence of delayed graft function, the duration of delayed graft function, the primary non - function rate, or the organ function at various time points after transplantation.

31. A method for assessing the outcome of a transplantation, the method comprising: preparing a nucleic acid preparation according to the method of any one of claims 1 to 23; and assessing the outcome of the transplantation based on at least a portion of the fragment size distribution of the nucleic acid.

32. The method according to claim 31, wherein the predicted outcome is at least partially predicted based on a risk determination generated by an algorithm that uses the fragment size distribution or a subset of the fragment size distribution as an algorithm input.

33. The method according to any one of claims 24 to 32, wherein the predicted outcome is rejection of the transplantation or non - rejection of the transplantation.

34. The method according to any one of claims 24 to 33, wherein the predicted outcome includes the type of transplantation rejection.

35. The method according to any one of claims 24 to 34, wherein the predicted outcome includes the time of transplantation rejection.

36. The method according to any one of claims 24 to 35, further comprising: making a recommendation to stop perfusion, pause transplantation, or delay transplantation of the organ or tissue based on the predicted outcome.

37. The method according to claim 36, wherein the recommendation is made if the amount of the nucleic acid exceeds the amount of a threshold nucleic acid.

38. The method according to any one of claims 1 to 37, further comprising: performing targeted genetic analysis on the isolated nucleic acid to identify genetic characteristics in the nucleic acid.

39. The method according to claim 38, wherein the targeted genetic analysis includes targeted amplification and high - throughput sequencing of at least 50 target loci in the nucleic acid.

40. The method according to any one of claims 38 to 39, further comprising: evaluating changes in homeostasis or cellular processes based on the targeted genetic analysis.

41. A method for preparing a non - naturally occurring nucleic acid preparation from a perfusion fluid or a flushing fluid, the nucleic acid preparation being useful for assessing the outcome of transplantation of a donor organ or donor tissue, the method comprising: obtaining a sample of the perfusion fluid or flushing fluid from a donor organ or donor tissue that has been perfused with the perfusion fluid or prepared with the flushing fluid, the perfusion fluid or flushing fluid comprising nucleic acids; isolating nucleic acids from the sample; and preparing the isolated nucleic acids for determination of at least one of the nucleic acid type in the sample or the sequence of the nucleic acids in the sample.

42. The method according to claim 41, wherein the isolated nucleic acid is RNA.

43. The method according to claim 42, wherein the nucleic acid type is coding RNA or non - coding RNA.

44. The method according to claim 43, further comprising: if the nucleic acid type is non - coding RNA, evaluating the expression regulatory function of the isolated nucleic acid.

45. The method according to claim 42, wherein high-throughput sequencing is used to evaluate the sequence.

46. The method according to any one of claims 42 to 45, wherein isolating the RNA comprises lysing vesicles containing the RNA.

47. A method for feedback-controlled mechanical perfusion of a donor organ or donor tissue, comprising: perfusing the donor organ or donor tissue in a perfusion chamber containing the donor organ or donor tissue at a first value of perfusion parameters; performing the method according to any one of claims 1 to 42 on the donor organ or donor tissue; generating an appropriate adjustment to the perfusion parameters based at least in part on a measurement of the isolated nucleic acid; and adjusting the perfusion parameters to a second value based on the generated appropriate adjustment.

48. The method according to claim 47, wherein the adjustment is an increase or decrease in the perfusion flow rate.

49. The method according to claim 48, wherein the perfusion flow rate is decreased to a minimum threshold perfusion rate required to maintain the transplant in a state evaluated to have a threshold likelihood of transplant rejection.

50. The method according to claim 49, wherein the threshold likelihood is the maximum likelihood of transplant rejection.

51. The method according to any one of claims 47 to 50, wherein the perfusion parameter is the concentration of at least one component of the perfusion fluid.

52. The method according to claim 51, wherein the at least one component is selected from the group consisting of oxygen, stem cells, immunosuppressive drugs, nutrients, or red blood cells.

53. The method according to any one of claims 47 to 52, wherein the perfusion parameter is the temperature or pH of the perfusion fluid.

54. The method according to any one of claims 47 to 53, further comprising: collecting a sample of the adjusted perfusion fluid; and evaluating whether additional adjustment is needed.

55. The method according to any one of claims 1 to 54, wherein the donor organ is a kidney, lung, heart, liver, gallbladder, pancreas, or intestine.

56. The method according to any one of claims 1 to 54, wherein the donor tissue is a heart valve, skin tissue, bone tissue, tendon, cornea, blood vessel, cartilage tissue, ligament, eye tissue, or bone marrow tissue.

57. The method according to any one of claims 1 to 54, wherein the donor tissue comprises umbilical cord blood stem cells or peripheral blood stem cells.

58. The method according to any one of claims 1 to 54, wherein the donor tissue is blood or platelets.

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