Nucleic acid analysis of perfusate or flush fluid

Abstract

Provided herein are systems and methods for preparing non-native DNA preparations from perfusate or flush, which preparations are useful for evaluating the transplant outcomes of donor organs or tissues perfused with perfusate or prepared for transplantation with flush. The preparations may also be useful for assessing the quality of the organ or tissue. Analysis of the DNA may be used to make clinical decisions. DNA analysis may also be used for adaptive control of machine perfusion systems used to preserve organs or tissues by generating and implementing appropriate parameter adjustments based on the characteristics of the sampled DNA.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 413,834, filed October 6, 2022, which is incorporated herein by reference in its entirety.

[0002] In the field of organ transplantation, controlled preservation of organs is often required after removal from the donor and before transplantation into the recipient to keep them viable and prevent organ damage and graft failure. Common preservation methods include the standard of care, simple cold storage (SCS), and various dynamic perfusion techniques. Dynamic organ perfusion attempts to mimic the physiological environment of the body by passing fluid through the organ in a cyclical manner.

[0003] Due to improved outcomes, machine perfusion is increasingly being used instead of SCS, especially for high-risk or marginal organs, such as kidneys recovered from post-mortem donors. SCS exposes organs to hypoxic conditions, which leads to the accumulation of metabolites from anaerobic metabolism. This accumulation of metabolites can increase the risk of ischemia-reperfusion injury (IRI) upon reperfusion in the recipient. Therefore, it is desirable to use a preservation method that not only regulates metabolite levels within the organ but also resupplies it with oxygenated blood / oxygen carriers, drugs, and essential nutrients. Furthermore, normothermic machine perfusion allows for the reconditioning of donor organs before transplantation, thus allowing for earlier restoration of blood flow and improving recipient outcomes. However, perfused organs can still sustain damage that adversely affects transplant outcomes, so methods for assessing the quality or condition of organs during perfusion before transplantation are needed.

[0004] Additionally, all solid organs preserved using SCS are flushed with crystalloid fluid in the operating room before transplantation. The purpose of this flush is to remove blood clots and other organic debris from the donor organ's circulatory system before anastomosis to the recipient's circulatory system. Summary of the Invention

[0005] The systems, devices, and methods described herein enable the analysis of nucleic acids sampled from perfusate, the fluid used for machine perfusion of an organ or tissue, or from organ flushes received from a donor. Both fluids are used to preserve and / or recondition organs / tissues during storage or transport prior to transplantation into a recipient; however, perfusate can be sampled according to the methods described herein to isolate and analyze DNA. Because perfusate and flush fluids are typically discarded, sampling, manipulation, and analysis of these fluids is not routine or conventional. Nucleic acids from perfusate, such as cell-free DNA (cfDNA), cellular DNA, or RNA, can be quantified, enriched to generate non-native preparations for analytical purposes, sequenced, amplified to generate non-native preparations for analytical purposes, or otherwise manipulated to generate non-native preparations for analytical purposes to inform physicians of the current status of the perfused organ / tissue. Analytical results from such preparations, such as cfDNA concentration or fragment size distribution (or a subset thereof), may indicate that the organ / tissue may be damaged and no longer suitable for transplantation, or that adjustments to the perfusion may be necessary. Analytical results obtained from such preparations may also be indicative of the quality of the donor organ / tissue, which can be used to assess suitability for transplantation and inform the process of allocating the organ / tissue to a suitable recipient. Analytical results may be predictive of early graft failure after organ / tissue transplantation, predictive of delayed graft function in kidney transplant recipients, and / or predictive of overall organ function after transplantation.

[0006] In a first aspect, provided herein is a method for preparing a nucleic acid preparation from perfusate useful for predicting transplant outcome of a donor organ or tissue, the method comprising obtaining a sample of perfusate from a donor organ or tissue perfused with perfusate, the perfusate containing nucleic acid, isolating the nucleic acid from the sample, and performing analysis of the isolated DNA to assess at least one of the amount of nucleic acid in the perfusate, the molecular weight of the DNA in the perfusate, or the fragment size distribution of the nucleic acid in the perfusate.

[0007] In some implementations, the amount of nucleic acid is the total amount of nucleic acid in the perfusate. In some implementations, the amount of nucleic acid is the amount of high molecular weight DNA (e.g., greater than 200 base pairs) in the perfusate. In some implementations, the amount of nucleic acid is the amount of low molecular weight DNA (e.g., less than 200 base pairs) in the perfusate. In some implementations, the molecular weight of the nucleic acid is the average molecular weight of the nucleic acid in the perfusate.

[0008] In some embodiments, the isolated nucleic acid is cell-free DNA. The method may further include assessing the cell-free DNA as 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 may be RNA present in extracellular vesicles such as exosomes and microvesicles. The captured vesicles can be lysed from the perfusate 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 assessing the quantity of the isolated RNA as a biomarker of organ / tissue stability or quality. The method may further include assessing a gene expression profile corresponding to the isolated RNA to assess the cellular origin of the RNA. For example, if the gene expression profile is associated with immune function, the cellular origin of the RNA is assessed as immune cells. RNA has the potential to be used as a biomarker, but it may also provide a more detailed picture of its origin (e.g., a more detailed signature of cell viability or organ status) when packaged in vesicles compared to free RNA. Due to the role of RNA in gene expression, analysis of RNA may provide more functional information about the state of an organ or tissue compared to analysis of DNA. Isolated RNA can be measured to assess at least one of the RNA type (e.g., coding or noncoding) or RNA sequence (e.g., mRNA sequence). The type and sequence(s) may provide information indicating the origin of the RNA found in the perfusate and may help provide information about the state of the organ or tissue. Because noncoding RNA can function as a modulator of expression, the presence or absence of noncoding RNA may be a predictor of transplant outcome and may be considered in addition to or instead of DNA or coding RNA. Manipulation and analysis of RNA can be more challenging than DNA due to the instability of RNA (due to single-stranded forms and uracil degradation) and lower natural RNA concentrations.Therefore, additional techniques may be required to accurately analyze RNA from perfusate or flush samples. For example, given the lower concentration of RNA, more selective isolation and extraction methods may be required. To address instability issues, RNA may be reverse transcribed to generate complementary DNA (cDNA).

[0009] The method may further include normalizing the amount of nucleic acid to at least one of the size, weight, volume, or surface area of ​​the donor organ or tissue, the perfusion time, the perfusion volume, or the temperature of the perfusion fluid. The method may include evaluating the fragmentation pattern of the nucleic acid. For example, the method may include classifying the isolated nucleic acid into nucleic acid derived from random degradation of ruptured cells in the perfusion fluid and nucleic acid derived from apoptosis of cells in the donor organ or tissue. The method may further include obtaining one or more additional samples of the perfusion fluid taken at different time points from the sample, quantifying the amount of nucleic acid (e.g., cellular DNA, cfDNA, or RNA) in the perfusion fluid in the one or more additional samples, and tracking the amount of nucleic acid in the fluid perfusion 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.

[0010] In a second aspect, provided herein is a method of predicting a transplant outcome, the method comprising preparing a preparation of nucleic acid according to the method of the first aspect above, and further comprising assuming a predicted outcome of transplantation of a donor organ or donor tissue based on the amount of nucleic acid.

[0011] In some embodiments, the nucleic acid is cellular DNA from cells derived from the donor, and the predicted outcome is a predicted good or bad prognosis based on the cellular DNA, for example, the cellular DNA indicating an immune response. In some embodiments, the nucleic acid is cell-free DNA, and the predicted outcome is a predicted good or bad prognosis based on the cell-free DNA, for example, the cell-free DNA indicating damage to the donor organ or tissue. In some embodiments, the predicted outcome is a predicted bad prognosis, which is a determining factor for not using the organ for transplantation. In some embodiments, the predicted outcome is assumed based at least in part on a risk call generated by an algorithm using the amount of nucleic acid as an algorithm input. In some embodiments, the predicted outcome is assumed by performing a "quality" assessment of the donor organ or tissue based on the amount of nucleic acid. Such an assessment may indicate at least one of the following characteristics associated with transplant outcome: the presence of delayed graft function, the duration of delayed graft function, the rate of early failure, and organ function at various time points after transplantation.

[0012] In a third aspect, provided herein is a method of predicting a transplant outcome, the method comprising preparing a nucleic acid preparation according to the method of the first aspect above, and further comprising assuming a predicted outcome of transplantation of a donor organ or tissue based on the fragment size distribution (or a subset thereof) of the nucleic acid. In some implementations, the predicted outcome is assumed based at least in part on a risk call assessed via an algorithm that uses the fragment size distribution (or a subset thereof) as an algorithm input.

[0013] In some implementations of either the second or third aspect, the predicted outcome is transplant rejection or non-rejection of the transplant. The predicted outcome may include one or more of the type of transplant rejection and the timing of transplant rejection. The method of either the second or third aspect may further include recommending stopping perfusion of the organ or tissue based on the predicted outcome. The recommendation to stop perfusion may be made if the amount of nucleic acid analyzed in one or more preparations exceeds a threshold amount of nucleic acid (e.g., a threshold calculated based on the size of the organ / tissue).

[0014] In some implementations of any of the first, second, or third aspects, the method further comprises performing targeted genetic analysis of the nucleic acids isolated in one or more preparations to identify genetic characteristics of the nucleic acids. Targeted genetic analysis can include targeted amplification and high-throughput sequencing of at least 50 target loci within the nucleic acids. Targeted genetic analysis can be used to evaluate changes in organ / tissue homeostasis or cellular processes.

[0015] In a fourth aspect, a method for feedback-controlled machine perfusion of a donor organ or donor tissue is provided, the method comprising perfusing the donor organ or donor tissue in a perfusion chamber holding the donor organ or donor tissue at a first value of a perfusion parameter, performing the method of any of the first, second, or third aspects on the donor organ or donor tissue, generating appropriate adjustments to the perfusion parameters based at least in part on the amount of nucleic acid in the perfusate, and adjusting the perfusion parameters to a second value based on the generated appropriate adjustments.

[0016] 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 flow rate necessary to maintain the transplant at an assumed or predicted threshold likelihood of transplant rejection. The threshold likelihood may be a 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, immunosuppressants, nutrients, or red blood cells. In some embodiments, the perfusion parameter is the temperature or pH of the perfusion fluid.

[0017] The method may further include taking a post-adjustment sample of the perfusate and assessing whether further adjustment is required.

[0018] In any of the above embodiments, the donor organ may be a kidney, lung, heart, liver, gallbladder, pancreas, intestine, or other organ. The donor tissue may be or include a heart valve, skin tissue, bone tissue, tendon, cornea, blood vessel, cartilage tissue, ligament, eye tissue, bone marrow tissue, or other tissue. The donor tissue may include blood, platelets, umbilical cord blood stem cells, or peripheral blood stem cells. Those skilled in the art will recognize that any donor tissue or graft may be subject to the described and claimed methods, and that the above donor tissues are exemplary and not limiting.

[0019] The foregoing and other objects and advantages will become apparent from the following detailed description considered in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout and in which: [Brief explanation of the drawings]

[0020] [Figure 1] 1 shows a block diagram of a machine perfusion system for storing and / or transporting donor organs or tissues, according to an exemplary implementation. [Figure 2] 1 shows a flowchart illustrating a method for preparing a nucleic acid preparation from a perfusate, according to an exemplary implementation. [Figure 3] 1 shows a flowchart illustrating a method for predicting transplant outcome by perfusate nucleic acid analysis, according to an exemplary implementation. [Figure 4] 1 shows a flowchart illustrating a method for feedback-controlled machine perfusion of a donor organ or tissue, according to an exemplary implementation. [Figure 5A] DNA size distribution of cell-free DNA isolated from perfusate samples is shown. [Figure 5B] DNA size distribution of cellular DNA isolated from perfusate samples is shown. [Figure 6A] DNA size distribution of cell-free DNA isolated from perfusate samples is shown. [Figure 6B] DNA size distribution of cellular DNA isolated from perfusate samples is shown. [Figure 6C] The DNA size distribution, including nucleosomal DNA, of a particular sample is shown. [Figure 7] 1 shows a plot of cellular DNA yield versus cell-free DNA yield from perfusate samples. [Figure 8] A graph of the percentage of cell-free DNA yield versus kidney weight for each perfusate sample is shown. [Figure 9A] A plot of cell-free DNA yield versus perfusion time is shown. [Figure 9B] A plot of cellular DNA yield versus perfusion time is shown. DETAILED DESCRIPTION OF THE INVENTION

[0021] To provide an overall understanding of the systems, methods, and devices described herein, several exemplary embodiments will be described. While the embodiments and features described herein are specifically described for use in connection with machine perfusion of organs and tissues, it will be understood that all components and other features outlined below can be combined with each other in any suitable manner and adapted and applied to other types of transplantation and / or perfusion methods. For example, fluid samples can be taken from organs or tissues undergoing simple cold storage (SCS) or cryopreservation, or from fluids used to flush organs or tissues prior to transplantation.

[0022] The systems, devices, and methods described herein allow for the generation and subsequent analysis of nucleic acid preparations from nucleic acids sampled from perfusate, the fluid used for machine perfusion of organs or tissues received from a donor, or from flush, the fluid used to prepare organs or tissues prior to transplantation. While machine perfusion is used to preserve or recondition organs / tissues during storage or transport before transplantation into a recipient, perfusate may also be sampled for nucleic acid isolation and analysis according to the methods described herein. Similarly, flush can be sampled and analyzed during or after organ or tissue preparation for transplantation. Nucleic acids from perfusate, such as cell-free DNA (cfDNA), cellular DNA, or RNA, can be quantified, enriched to generate non-native preparations for analysis, sequenced, amplified to generate non-native preparations for analysis, or otherwise manipulated to generate non-native preparations for analysis to inform physicians of the current status of the perfused organ / tissue. Analytical results, such as cfDNA concentration or fragment size distribution (or a subset thereof), may indicate that the organ / tissue may be damaged and no longer suitable for transplantation, or that adjustments to perfusion may be necessary. The analytical results can also indicate the quality of the donor organ / tissue, which can be used to assess suitability for transplantation and inform the process of allocating organs / tissues to suitable recipients. Levels of nucleic acids, e.g., cfDNA, in the perfusate can predict primary graft dysfunction after organ / tissue transplantation.

[0023] It will be understood that the systems and methods described herein can be applied to any organ, tissue, or biological entity that is perfused or fluid-treated before or during transplantation. The present disclosure is applicable to, but not limited to, kidney, lung, heart, liver, gallbladder, pancreas, intestine, heart valve, skin tissue, bone tissue, tendon, cornea, blood vessel, cartilage tissue, ligament, eye tissue, or bone marrow tissue, or combinations thereof. In some implementations, the organ / tissue of interest is donated blood, donated platelets, umbilical cord blood stem cells, or peripheral blood stem cells, any of which can be stored in a fluid that can be sampled for nucleic acid analysis. These organs, tissues, or biological entities can be perfused and / or sampled before or after organ recovery from the donor. The term "graft" may be used to describe one or more of the organs or tissues described herein, and transplants suitable for these methods and systems include, but are not limited to, autografts, syngrafts, allografts, and xenografts.

[0024] While the examples of this disclosure discuss various techniques for generating DNA preparations, manipulating DNA, and analyzing DNA, those skilled in the art will understand that this disclosure can be extended to the manipulation and analysis of RNA or other nucleic acids present in perfusate or flush samples. While the examples provided herein may refer only to perfusate, it is understood that these methods can be applied to flush samples without departing from the scope of this disclosure. Both perfusate and flush fluids are sometimes referred to herein as "graft fluid." The graft fluid may include an oxygen carrier, a buffer or priming solution, colloids, nutritional supplements, anticoagulants, protective additives, and / or antibiotics. The graft fluid may be a red blood cell-based solution, acellular solution, or whole blood. The oxygen carrier may include red blood cells. In some embodiments, the oxygen carrier is a hemoglobin-based oxygen carrier, such as Hemopure or Breonics. Hemopure is a polymerized bovine hemoglobin-based oxygen carrier. Breonics is an extravascular metabolic support system containing a highly concentrated tissue-culture-like medium containing amino acids, lipids, carbohydrates, and bovine hemoglobin. The acellular graft solution may be LIFOR®, a non-protein oxygen carrier; Aqix RS-I®, primarily used for tissue biopsy preservation; or STEEN® solution, which contains high concentrations of albumin and dextran. STEEN® solution may be diluted, particularly for use in kidney perfusion. The graft solution may contain an agent to prevent cellular edema, such as albumin. The graft solution may also contain an agent to increase osmolality and / or promote blood flow, such as mannitol. The graft solution may also contain a vasodilator, such as prostacyclin. The prostacyclin may be a synthetic form, such as epoprostenol. The vasodilator may also be verapamil or sodium nitroprusside. The graft solution may also contain an agent to prevent inflammation, such as a corticosteroid. The graft fluid may contain nutrients such as glucose, amino acids, insulin, and / or multivitamins. The graft fluid may also contain antibiotics.The graft solution may include a priming solution such as RINGER'S® solution, STEEN® solution, Plasma Lyte A, or Williams media E. In one preferred embodiment, the graft solution is a combination of STEEN® solution, either acellular or with packed RBCs added to the perfusate, optionally with autologous whole blood added to STEEN® in place of RBCs. Composition of STEEN® solution: (1000 ml contains 5 g dextran 40 [0.125 mM], 70 g bovine serum albumin [1.05 mM], 5.03 g NaCl [85.90 mM], 0.24 g glucose monohydrate [1.21 mM], 0.34 g KCl [4.56 mM], 0.19 g NaH2PO4·2H2O [1.22 mM], 0.22 g CaCl2·2H2O [1.50 mM], 0.24 g MgCl2·6H2O [2.52 mM], 1.26 g NaHCO3 [15.00 mM], adjusted to pH 7.4 with 1 M NaOH). Table 2 below contains further exemplary graft fluids disclosed in Elliott et al., Am. J. of Transpl. 21(4), 1382-1390 (2021), which is incorporated herein by reference. [Table 1-1] [Table 1-2]

[0025] mechanical perfusion FIG. 1 illustrates an exemplary machine perfusion system 100 for storing, preserving, transporting, and / or reconditioning a donor organ or tissue 104 for transplantation into a recipient. The system 100 includes a chamber 102 for holding the organ / tissue 104. Perfusate is delivered into and out of the chamber 102 via a conduit assembly 106 that fluidly connects the chamber 102 to other components of the system 100, such that the conduit assembly 106 and the components define a fluid circuit. The components include a reservoir 108 for holding perfusate directed away from the chamber 102. An inlet 110 is coupled to the reservoir 108. The components further include a pump 112 for driving the perfusate through the fluid circuit via the conduit assembly 106, an oxygenator 114 for oxygenating the perfusate, a heat exchanger 116 for cooling or heating the perfusate, and one or more sensors 118 for measuring one or more properties of the perfusate. A separator 122 is provided within (or otherwise operably coupled to) chamber 102 for removing one or more substances from the chamber or perfusate. Controller 120 is operably coupled to each of inlet 110, pump 112, oxygenator 114, heat exchanger 116, and sensor(s) 118 such that controller 120 can send and / or receive data from each component.

[0026] As described in further detail below, methods for analyzing the perfusate can be performed after sampling the perfusate from system 100. The results of the analysis can be used to inform feedback control of system 100 or for a physician or operator to manually adjust certain parameters of system 100. While the components of system 100 are shown connected in a particular order in a fluid circuit via conduit assembly 106, it should be understood that the components can be rearranged in any suitable order. Two or more components can be arranged in series or in parallel, with the latter configuration causing the conduit assembly to split into two or more flow paths for delivering perfusate through each of the two or more components. Certain components may be omitted.

[0027] It should be understood that the organ / tissue 104 can be any biological entity that requires or benefits from perfusion, for example, for preservation, storage, transportation, and / or reconditioning, including, for example, a kidney, lung, heart, liver, gallbladder, pancreas, intestine, heart valve, skin tissue, bone tissue, tendon, cornea, blood vessel, cartilage tissue, ligament, eye tissue, bone marrow tissue, a volume of blood, a volume of platelets, a volume of umbilical cord blood stem cells, or a volume of peripheral blood stem cells.

[0028] The perfusion fluid delivered through the fluid circuit of the system 100 may be any perfusion fluid used in graft preservation or preparation. For example, the perfusion fluid may be or include a solvent such as water containing a combination of proteins, sugars, and / or soluble salts. In some implementations, the perfusion fluid includes red blood cells. The perfusion fluid may be a sterile, isotonic solution. For example, KPS-1® Kidney Perfusion Solution (Organ Recovery Systems, Itasca, IL) may be used for machine perfusion of the kidney. A suitable perfusion fluid may be or include a sodium lactate solution (also known as Ringer's lactate solution or Hartmann's solution). The perfusion fluid may include at least one of sodium chloride, sodium lactate, potassium chloride, calcium chloride, magnesium sulfate, mannitol, dexamethasone, glutathione, or insulin. In either implementation, the perfusate within the chamber 102 and the perfusate transported from the chamber 102 by the conduit assembly 106 contains nucleic acids from the organ / tissue 104. The nucleic acids in the perfusate may be present as cellular DNA, cell-free DNA, or RNA, or a combination thereof.

[0029] The system 100 may include a housing in which one or more components are enclosed. For example, all components may be enclosed within the housing. The housing may include one or more ports for connection to an external source or drain, for example, for the inlet 110 or the separator 122. The housing may be a transporter equipped with a carrying handle for transporting and storing the donor organ / tissue 104. Alternatively, the system 100 does not include a housing. The components may be loosely arranged and connected via electrical leads for the conduit assembly 106 and the controller 120. In some implementations, the chamber 102 is at least part of a cadaver, and the organ / tissue 104 has not yet been removed from the cadaver. The remainder of the system 100 may be enclosed within a housing that is fluidly connected to the cadaver via the conduit assembly 106.

[0030] The chamber 102 is a container for maintaining the organ / tissue 104 in a sterile condition during storage or transport, typically for preserving and / or reconditioning the organ / tissue 104. The chamber 102 may be a container having walls, an inlet, and an outlet, which are fluidly coupled to a conduit assembly 106 for transporting perfusate into and out of the chamber 102. The chamber 102 may be a sterile container and / or a disposable container. The chamber 102 may be constructed of a biocompatible material, such as a thermoplastic elastomer (TPE). The chamber 102 may include a lid that allows access to the chamber 102 and the organ / tissue 104. The chamber 102 may have a rigid, flexible, or foldable structure. In some implementations, the chamber 102 includes one or more measurement ports, and sensor(s) 118 may be inserted into the chamber 102 via the one or more measurement ports to measure properties of the perfusate, properties of the organ / tissue 104, and / or conditions of the chamber 102. Examples of measurements are described in more detail below.

[0031] The conduit assembly 106 may be formed from any suitable tubing material, such as a biocompatible plastic. The conduit assembly 106 may be rigid or flexible. In some implementations, electrical leads are attached to or formed on the conduit assembly 106 to connect the controller 120 to either the pump 112, the oxygenator 114, the heat exchanger 116, or the sensor(s) 118.

[0032] Reservoir 108 receives perfusate delivered from chamber 102 via conduit assembly 106. Reservoir 108 may be a holding chamber with a perfusate inlet and a perfusate outlet, each coupled to conduit assembly 106 for delivering perfusate. Inlet 110 is coupled to reservoir 108 for introducing substances such as new perfusate, new perfusate components, therapeutic agents, nutrient supplies, stem cells, and / or blood cells. Inlet 110 is operatively coupled to controller 120, which allows substances to be added to the perfusate in response to user input to controller 120 or in a feedback control loop implemented by controller 120, as discussed in more detail below. In some implementations, separator 122 is connected to or disposed within reservoir 108 to remove certain substances from the perfusate, as discussed in more detail below.

[0033] The pump 112 drives the flow of perfusion fluid through the fluid circuit of the system 100. The pump 112 may be any suitable type of fluid pump, such as a non-positive displacement (e.g., centrifugal) or positive displacement (e.g., peristaltic, plunger, or piston) pump. The pump 112 may induce a continuous or pulsatile flow of perfusion fluid. The pump 112 is operatively coupled to the controller 120, which may utilize user input or feedback control to set the flow rate of the pump 112. The flow rate may be set per mass of the organ / tissue 104.

[0034] The oxygenator 114 supplies oxygen to the perfusate. The oxygenator 114 may include a port for accepting oxygen from an external source, such as an oxygen concentrator, an oxygen tank, or wall oxygen. Alternatively, the oxygenator 114 may include an oxygen tank or an oxygen concentrator. For example, the oxygenator 114 may be an oxygen concentrator, including an air intake for accepting ambient air, which is then concentrated to produce a flow of oxygen-enriched air for oxygenating the perfusate.

[0035] The heat exchanger 116 is configured to cool or heat the perfusate. Perfusion of the organ / tissue 104 using the system 100 can be performed at different temperatures ranging from hypothermia (4-10°C) to sub-normothermia (15-30°C) to normothermia (approximately 37°C). Thus, the heat exchanger 116 may be used to maintain a target temperature of the perfusate (e.g., 0-40°C) by heating or cooling. The controller 120 is operatively coupled to the heat exchanger 116 to set the target temperature and / or adjust the temperature of the perfusate. The controller 120 may accept user input to set or adjust the target temperature, which is then controlled to reach the target temperature. In some implementations, a feedback control method is used to adjust the temperature of the perfusate. The heat exchanger 116 may include a temperature sensor, or sensor(s) 118 may measure the temperature and communicate the measured temperature to the controller 120 for appropriate control of the heat exchanger 116.

[0036] The sensor(s) 118 are configured to measure one or more properties of the perfusate. The properties measured by the sensor(s) 118 may include at least one of temperature, flow rate, pH, oxygen concentration, nucleic acid concentration, toxin concentration, or the concentration of another perfusate component. The values ​​of the measured properties are communicated by the sensor(s) 118 to the controller 120. The measurements may be used for feedback control of one or more components of the system 100 or to inform clinical decision making.

[0037] Separator 122 can be used to remove materials from chamber 102. While separator 122 is shown in FIG. 1 as adjacent to or attached to chamber 102, it should be understood that the separator or outlet may be located anywhere in the fluid circuit of system 100, such as within conduit assembly 106 or reservoir 108. For example, separator 122 can be a filter (e.g., a micropore filter, an ultrafiltration device), a separation column, a precipitated material trap, or a collection of probes (e.g., affinity probes, magnetic probes). Materials such as metabolites removed by separator 122 can be flushed from system 100.

[0038] The controller 120 is, for example, a microcontroller, processor, or 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 perfusion based on measured characteristics of perfusion or by receiving input of stability or damage characteristics of the organ / tissue 104. Such input can be results generated according to the analysis methods described herein.

[0039] Perfusate sample preparation method As described above, perfusate from a machine perfusion system such as system 100 may be sampled, and nucleic acids within the perfusate sample may be manipulated, amplified, prepared, and / or analyzed according to methods described herein. Figures 2-4 describe methods for performing and utilizing this nucleic acid analysis.

[0040] FIG. 2 shows a flowchart illustrating an exemplary method 200 for preparing a nucleic acid sample from perfusate used in the preservation and / or reconditioning of a donor organ / tissue. This method is particularly useful for assessing or predicting the quality of a donor organ / tissue for transplantation and the outcome of the donor organ / tissue's subsequent transplantation. Method 200 includes steps 202, 204, and 206. Step 202 involves obtaining a sample of perfusate from a donor organ / tissue or a machine perfusion system, such as system 100 described above in connection with FIG. 1. The sample includes nucleic acid. Step 204 involves isolating nucleic acid from the sample. Step 206 involves performing analysis of the isolated DNA preparation to assess at least one of the amount of nucleic acid in the perfusate, the molecular weight of the nucleic acid in the perfusate, or the fragment size distribution of the nucleic acid in the perfusate.

[0041] The nucleic acids isolated from the sample in step 204 may be cellular DNA, cell-free DNA (cfDNA), or RNA. Thus, cellular DNA, cfDNA, or RNA can be separated from the perfusate in the sample using isolation techniques, including, but not limited to, centrifugation, size selection, hybrid capture, or other suitable techniques. In some embodiments, the sample may be centrifuged to separate the various layers. In some embodiments, nucleic acids may be isolated using filtration. In some embodiments, nucleic acid preparation may involve amplification, separation, chromatographic purification, liquid-liquid separation, preferential enrichment, preferential amplification, target amplification, reverse transcription, or any of the many other techniques described herein, or any combination thereof. In some embodiments for DNA isolation, RNase is used to degrade RNA. Alternatively, DNase may be used to degrade DNA for the purpose of isolating RNA. Suitable purification techniques further include, but are not limited to, differential ultracentrifugation, density gradient ultracentrifugation, polymer-facilitated precipitation, immunoaffinity capture, and size exclusion chromatography. Those skilled in the art will recognize that the aforementioned isolation techniques are exemplary and not exhaustive. The aforementioned or other isolation techniques may be integrated with measurement, quantification, and multi-omics characterization techniques on a microfluidic platform.

[0042] For example, centrifugation may be used to separate the cell pellet from the perfusate. Cellular DNA may be purified from the cell pellet using methods or devices, including but not limited to, cellular DNA-specific probes, size selection, or selectively binding silica-based membranes, and cfDNA may be purified from the remaining perfusate using methods or devices, including but not limited to, cfDNA-specific probes, size selection, or circularized DNA systems. Nucleic acids may be isolated from the cellular source by various extraction methods. These methods involve lysing the cells, thereby liberating the nucleic acids and leaving the chromatin structure sufficiently intact to allow for the preparation of a nucleosome ladder, i.e., a nucleosome preparation. Suitable cell lysis methods include methods that separately release nuclei for subsequent isolation and methods that dissolve the nuclear membrane. In some embodiments, cells may be permeabilized using detergents, such as lysolecithin, to preserve chromatin structure. In some embodiments, cell membranes may be disrupted by inducing apoptosis in the source cells. It is important to prepare nucleic acids free of other cellular components to enable biochemical manipulation of the nucleosome ladder for use in subsequent procedures, such as DNA sequencing. In one embodiment, the nucleic acid system can be used to purify cellular DNA. For example, cfDNA can be isolated from the remaining perfusate using beads with specific surface chemistries.

[0043] Techniques such as amplification may be used between steps 204 and 206 to further prepare, modify, purify, and / or enrich the isolated nucleic acids. In some embodiments, universally tagged adapters are added to create a library. In some implementations, the tagged adapters can be added using PCR. In some implementations, the tagged adapters can be added using ligation. Prior to ligation, the sample nucleic acids are blunt-ended and then a single adenosine base is added to the 3' end. Prior to ligation, the nucleic acids can be cleaved using a restriction enzyme or other cleavage method. During ligation, the 3' adenosine of the sample fragment and the complementary 3' tyrosine overhang of the adapter can increase ligation efficiency. In some embodiments, the library is amplified using universal primers. In one embodiment, the amplified library is fractionated by size separation or other methods. In some embodiments, PCR amplification is used to amplify the target loci. In some embodiments, the amplified nucleic acids are sequenced (e.g., 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 at a particular base when sequencing from one end of the amplified nucleic acid, there is less chance of a sequence error at 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 resequenced from one or both ends (or by increasing the sequencing cycle) to increase the "read depth." As used herein, the term "read depth" refers to the number of sequencing reads mapped to a particular locus. Read depth may be normalized across the total number of reads. When "read depth" refers to a sample, it can refer to the average read depth of the target locus. When "read depth" refers to a locus, it can refer to the number of reads measured by a sequencer mapped to that locus.In general, the deeper the read depth at a locus, the closer the allele ratio at that locus tends to be to the allele ratio in the original sample of DNA. In general, increasing read depth can reduce sequencing errors.

[0044] In some embodiments, whole genome amplification (WGA) is used to amplify nucleic acid samples. In one embodiment, WGA is performed using ligation-mediated PCR (LM-PCR), in which short DNA sequences called adapters are ligated to blunt ends of DNA. These adapters contain universal amplification sequences that are used to amplify DNA by PCR. In another embodiment, WGA is performed using degenerate oligonucleotide primer PCR (DOP-PCR), in which random primers that also contain universal amplification sequences are used in the first round of annealing and PCR. A second round of PCR is then used to further amplify the sequences using the universal primer sequences. In another embodiment, WGA is performed using multiple displacement amplification (MDA), which uses phi29 polymerase, a highly processive and nonspecific enzyme that replicates DNA and has been used in single-cell analysis. In some embodiments, WGA is not performed.

[0045] In some embodiments, selective amplification or enrichment is used to amplify or enrich target loci. In some embodiments, amplification and / or selective enrichment techniques may involve PCR, such as ligation-mediated PCR, fragment capture by hybridization, molecular inversion probes, or other circularization 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 are used (Hung et al., J Clin Pathol 62:308-313, 2009, incorporated herein by reference in its entirety). In some embodiments, hybridization capture using hybrid capture probes is used to preferentially enrich nucleic acids. In some embodiments, methods for amplification or selective enrichment may involve using probes that, when properly hybridized to the target sequence, separate the 3' or 5' end of the nucleotide probe from the polymorphic site of the polymorphic allele by a small number of nucleotides. This separation reduces preferential amplification of one allele, referred to as allelic bias. This is an improvement over methods involving the use of probes in which the 3' or 5' end of a correctly hybridized probe is located immediately adjacent to or very close to the polymorphic site of an allele. In one embodiment, probes whose hybridizing region may or definitely contains the polymorphic site are excluded. Polymorphic sites at the hybridization site may cause unequal hybridization or completely inhibit hybridization at some alleles, resulting in preferential amplification of certain alleles. These embodiments are an improvement over other methods involving target amplification and / or selective enrichment in that they better preserve the original allele frequencies of a sample at each polymorphic locus, whether the sample is a pure genomic sample from a single individual or a mixture of individuals.

[0046] In some embodiments, a PCR technique called mini-PCR is used to generate very short amplicons (see U.S. Application No. 13 / 683,604, filed November 21, 2012; U.S. Publication No. 2013 / 0123120; U.S. Application No. 13 / 300,235, filed November 18, 2011; U.S. Publication No. 2012 / 0270212, filed November 18, 2011; and U.S. Application No. 61 / 994,791, filed May 16, 2014, the contents of each of which are incorporated herein by reference in their entirety). cfDNA is highly fragmented. In some cfDNA, fragment sizes are distributed in an approximately Gaussian pattern, with a mean of 160bp, a standard deviation of 15bp, a minimum size of about 100bp, and a maximum size of about 220bp. The polymorphic site at a particular target locus may occupy any position from the beginning to the end of various fragments derived from that locus. Because cfDNA fragments are short, the likelihood that both primer sites are present, i.e., the likelihood that a fragment of length L contains both a forward primer site and a reverse primer site, is the ratio of the amplicon length to the length of the fragment. Under ideal conditions, assays with amplicons of 45, 50, 55, 60, 65, or 70 bp will successfully amplify 72%, 69%, 66%, 63%, 59%, or 56% of the available template fragment molecules, respectively. In certain embodiments, cfDNA is amplified using primers with melting temperatures of 50-65°C, and 54-60.5°C, resulting in a maximum amplicon length of 85, 80, 75, or 70 bp, or in certain preferred embodiments, 75 bp. The amplicon length is the distance between the 5' ends of the forward and reverse priming sites. Amplicon lengths shorter than those typically used may result in more efficient measurement of desired polymorphic loci by requiring shorter sequence reads, hi one embodiment, a substantial portion of the amplicons are less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp.

[0047] In some embodiments, the amplification is performed using direct multiplex PCR, sequential PCR, nested PCR, doubly nested PCR, one-and-a-half sided nested PCR, fully nested PCR, one sided fully nested PCR, one-sided nested PCR, hemi-nested PCR, hemi-nested PCR, triply hemi-nested PCR, semi-nested PCR, one-sided semi-nested PCR, reverse semi-nested PCR, or reverse nested PCR. Mini-PCR is performed using semi-nested PCR, or one-sided PCR, as described in U.S. Application No. 13 / 683,604, filed November 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed November 18, 2011, U.S. Publication No. 2012 / 0270212, and U.S. Application No. 61 / 994,791, filed May 16, 2014, which are incorporated by reference in their entireties. Any of these methods can be used for mini-PCR, if desired.

[0048] If necessary, the extension step of the PCR amplification can be time-limited to reduce amplification from fragments longer than 200, 300, 400, 500, or 1,000 nucleotides, potentially improving test performance by enriching for fragmented or short DNA (e.g., DNA from cells undergoing apoptosis or necrosis).

[0049] 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 generate a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to generate amplification products containing target amplicons. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplification products are primer dimers. In some embodiments, the primers are in solution (e.g., dissolved in a liquid phase rather than a solid phase). In some embodiments, the primers are in solution and not immobilized on a solid support. In some embodiments, the primers are not part of a microarray. In some embodiments, the primers do not comprise a molecular inversion probe (MIP).

[0050] In step 206, an analysis of the isolated nucleic acid preparation is performed to assess at least one of the amount of nucleic acid in the perfusate, the molecular weight of the nucleic acid in the perfusate, or the fragment size distribution of the nucleic acid in the perfusate. Various techniques can be used to perform this analysis. An appropriate technique can be selected by balancing the variations in accuracy, reproducibility, sensitivity, labor intensity, speed, and cost between the various techniques. Suitable techniques include, for example, the use of 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), GX TOUCH 24 HIGH SENSITIVITY ASSAY (PERKINELMER), or FRAGMENT ANALYZER HIGH SENSITIVITY NGS KIT (ADVANCED ANALYTICAL). Suitable methods / techniques include, but are not limited to, UV-vis spectrophotometry, fluorometry (e.g., fluorescent 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, one or more loci in cfDNA or cellular DNA (e.g., glyceraldehyde-3-phosphate dehydrogenase, GAPDH, etc.) can be measured using multiplex qPCR. In some embodiments, fluorescently labeled PCR is used to measure cfDNA, cellular DNA, or RNA. If desired, methods such as, but not limited to, the Shapiro-Wilk test can be used to calculate normal distribution of data. If desired, methods such as the Mann-Whitney U test can be used to compare levels of cfDNA, cellular DNA, or RNA.In some embodiments, methods such as the Mann-Whitney U test or the Kruskal-Wallis test are used to compare levels of cfDNA, cellular DNA, or RNA to other prognostic factors.

[0051] The amount of nucleic acid assessed in step 206 may be the total amount of nucleic acid in the perfusate sample, an estimate of the total amount of nucleic acid in the perfusate of a machine perfusion system, the concentration of nucleic acid in the perfusate 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 assessed in step 206 may be the average molecular weight of the nucleic acid in the perfusate or the molecular weight distribution of the nucleic acid.

[0052] Method 200 may include additional steps for interpreting or refining the analysis results of step 206. For example, if the isolated and analyzed nucleic acid is cfDNA, method 200 may further include assessing that the cfDNA is derived from apoptosis if the cfDNA has a molecular weight (assessed in step 206) consistent with nucleosomal DNA. The method may further include assessing a change in homeostasis or cellular process based on the analysis results. For example, elevated cfDNA levels in the perfusate may indicate that an organ / tissue is trending away from homeostasis and may be or has become dysfunctional.

[0053] In some implementations, the nucleic acid isolated in the preparation is RNA. The RNA may be RNA present in extracellular vesicles such as exosomes and microvesicles. Vesicles captured from the perfusate may be lysed to release the RNA, which may then be purified in solution using techniques such as filtration, size selection, or hybrid capture. The method may further include assessing the quantity of 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 assess the cellular origin of the RNA. For example, if the gene expression profile is related to immune function, the cellular origin of the RNA is assessed as immune cells. While RNA can be used as a biomarker like DNA, it may also provide a more detailed picture of its origin (i.e., a more detailed signature of cell viability or organ status) when packaged in vesicles compared to free RNA, for example. Due to the role of RNA in gene expression, analysis of RNA may provide more functional information about the state of an organ or tissue compared to analysis of DNA. Manipulating and analyzing RNA can be more challenging than manipulating and analyzing DNA due to RNA instability (due to its single-stranded nature, uracil degradation, and the ubiquitous presence of ribonuclease enzymes in cells and tissues that rapidly degrade RNA) and lower natural RNA concentrations. Therefore, additional techniques may be required to accurately isolate and analyze RNA from perfusate or flush samples. For example, more selective isolation and extraction methods may be used to account for the lower concentration of RNA. To improve stability, RNA can also be reverse transcribed to create complementary DNA (cDNA).

[0054] Given the instability and low abundance of RNA, suitable techniques for isolating RNA from a sample or preparation include, but are not limited to, guanidinium acid 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., hybrid capture with biotin-labeled probes and streptavidin-coated magnetic beads), lithium chloride and urea isolation, chromatography (e.g., oligo(dt)cellulose column chromatography), and non-column poly(A)+ purification / isolation. Suitable techniques may include cell lysis and digestion, DNA and protein denaturation, RNase denaturation and inactivation, removal or separation of cellular components, or precipitation. In some embodiments, RNA is reverse transcribed to produce cDNA, which is then analyzed in step 206. Results of the cDNA analysis can be traced back to characterize the RNA in the original sample or preparation.

[0055] In some implementations, analysis of RNA (e.g., mRNA) from the perfusate or flush is used to assess the cellular transcriptome(s) of the graft at any given moment. Thus, the method can include transcriptome assembly from sequencing reads of the RNA or corresponding cDNA (e.g., by microarray or RNA-Seq). For 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 performed de novo (without a reference genome) or genome-guided, eliminating the need for prior genotyping of the graft or donor. A de novo approach involves identifying contiguous sequences in the sequence reads (e.g., using a de Bruijn graph). A genome-guided approach can be used when the genome of the graft or donor is already known. Alternatively, DNA in the same or separate perfusate or flush samples can be genotyped in conjunction with RNA analysis to generate a reference genome in parallel. Genome-guided approaches align sequence reads on contiguous and non-contiguous sequences.

[0056] RNA analysis may also involve quantifying gene expression. Quantifying expression can be useful for studying responses to external stimuli, differences between healthy and diseased states, and other questions regarding graft stability. For example, changes in perfusion (or flush) conditions (e.g., temperature, pH, solute concentration) can alter gene expression within graft cells, and analysis of this dynamic expression can be used to provide information regarding adjustments to perfusion (or flush) conditions, as described below with respect to Figure 4. Expression can be quantified by counting the number of reads mapped to each locus during the transcriptome assembly step. Expression can be quantified for exons or genes, for example, using annotations of identified sequential or reference transcripts. The observed number of reads can be converted into appropriate metrics for hypothesis testing, regression, or other analyses, taking into account, for example, sequencing depth or coverage, gene length, total sample RNA (e.g., as assessed in step 206), or the variance in expression of each gene. Considering these parameters can help normalize results between samples and loci and reduce error (e.g., sampling error propagating through the analysis).

[0057] As discussed herein, multiple samples (of perfusate or flush) can be collected over time or for different grafts and subjected to method 200. RNA analysis from multiple samples can be particularly useful for assessing differential expression between two or more conditions (e.g., perfusion conditions such as temperature or pH) or between 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 the differential expression analysis performed on the multiple samples includes differentially expressed genes (DEGs) that may be up- or down-regulated. The differential expression analysis can take as input: (1) a gene expression matrix containing M genes for each of the N samples analyzed by gene expression quantification, and (2) a design matrix containing the experimental conditions for the N samples. Relevant conditions include (but are not limited to) physical conditions (e.g., temperature, pH, or concentration of perfusion or flush solutions, graft size), batch effects (e.g., laboratory conditions, measurement error, instruments or techniques used, reagent lots or batches, individual variability, time of day), genetic influences (e.g., known artifacts or variations), and / or any metadata that may alter gene expression. Conditions may be known or unknown, and unknown conditions can be estimated using machine learning approaches (e.g., principal components, surrogates, etc.). Hidden variable analysis may also be performed to identify uncaptured conditions. Expression variation analysis may involve regression or nonparametric statistics to identify DEGs. Adjustments such as familywise error rate or false discovery rate may be employed to account for multiple hypotheses. The output of expression variation analysis may include a table showing the log-fold change, p-value, and / or p-value adjusted for multiple comparisons for each gene. A cutoff for log fold change may be set to identify biologically relevant DEGs (those that pass the cutoff and are statistically significant).The identified biologically relevant DEGs can be used to inform decisions regarding graft(s). For example, if immune response-related genes are upregulated, the graft may require perfusion adjustment or treatment with drugs (e.g., immune stabilizers). Because specific genes may be identified as biomarkers for graft rejection or failure, the identified upregulation of those genes can serve as an indicator for predicting graft rejection or failure.

[0058] The results of step 206 may be normalized to account for specific parameters of the machine perfusion system from which the sample is taken. For example, the amount, molecular weight, or fragment size distribution of nucleic acids may be normalized with respect to one or more of perfusion time (the amount of time the organ / tissue is perfused), perfusate volume, organ / tissue size, organ / tissue weight, organ / tissue volume, organ / tissue surface area, perfusate temperature, and perfusate pH. Thus, each of these parameters can be measured before or during method 200.

[0059] Method 200 may further include assessing the fragmentation pattern of the nucleic acids, which can be used to classify the isolated nucleic acids into those derived from random degradation of ruptured cells in the perfusate and those derived from apoptosis of cells within the donor organ / tissue.

[0060] Additional samples of perfusate from the donor organ / tissue may be taken at various 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 time. By monitoring the amount, molecular weight, or fragment size distribution (or a subset thereof) of nucleic acid over time, a physician can closely monitor the quality or condition of the donor organ / tissue and assess whether intervention or adjustment is necessary, as described in more detail below.

[0061] FIG. 3 shows a flowchart illustrating a method 300 for predicting transplant outcomes. Method 300 includes steps 302, 304, 306, and 308. Step 302 involves obtaining a sample of perfusate from a donor organ / tissue or machine perfusion system, such as system 100 described above in connection with FIG. 1. The sample includes nucleic acids. Step 304 involves isolating the nucleic acids from the sample to create an artificial nucleic acid preparation. Step 306 involves performing an analysis of the isolated nucleic acid preparation to assess at least one of the amount of nucleic acid in the perfusate, the molecular weight of the nucleic acid in the perfusate, or the fragment size distribution of the nucleic acid in the perfusate. Steps 302, 304, and 306 can be performed in the same manner as described above in connection with steps 202, 204, and 206 of FIG. 2, respectively. Step 308 involves predicting the outcome of the transplant 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 of the nucleic acid (or a subset thereof).

[0062] Predicted outcomes may include the presence of delayed graft function, the duration of delayed graft function, the rate of early failure, organ function at various times after transplant, the likelihood of graft rejection, the likelihood of graft non-rejection, the type of graft rejection, or the timing of rejection.

[0063] The results of the analysis in step 306 can provide useful information regarding the quality or condition of the donor organ or tissue, or the clinical outcome of transplantation of the organ or tissue into a recipient. For example, if the isolated and analyzed nucleic acid preparation is cellular DNA from cells derived from the donor, the predicted result of step 308 can be a predicted good or bad prognosis based on the cellular DNA indicating an immune response by cells derived from the organ / tissue donor. As another example, if the isolated and analyzed nucleic acid is cfDNA, the predicted result can be a predicted good or bad prognosis based on the cfDNA indicating damage to the donor organ / tissue. The predicted result of step 308 can be used, for example, if the predicted result is a good or bad prognosis, to adjust perfusion parameters or to determine whether to reject the organ / tissue for transplantation. The predicted result can be used to make recommendations to adjust or discontinue perfusion of the organ / tissue. For example, a recommendation to discontinue perfusion is made if the estimated amount of nucleic acid in the perfusate exceeds a threshold amount of nucleic acid.

[0064] The outcome of step 308 can be predicted based, at least in part, on a risk call generated by an algorithm that uses the assessed nucleic acid quantity, molecular weight, or fragment size distribution (or a subset thereof) as algorithm input(s). The outcome can be predicted by performing a quality assessment of the donor organ / tissue based on the nucleic acid quantity, molecular weight, or fragment size distribution (or a subset thereof). Various methods can be used to perform the quality assessment. For example, to summarize the risk of kidney graft failure, a modified kidney donor risk index can be calculated incorporating the assessed nucleic acid quantity, molecular weight, or fragment size distribution (or a subset thereof). The quality assessment can involve scoring the graft based on nucleic acid analysis. Scoring can include, but is not limited to, calculating a graft-specific quality index (e.g., a liver graft quality index, a lung graft quality index, or a kidney donor risk index (KDPI)).

[0065] FIG. 4 shows a flowchart illustrating a method 400 for feedback-controlled machine perfusion of a donor organ or tissue. 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 a perfusion parameter. Step 404 involves obtaining a sample of perfusate from the donor organ / tissue or from a machine perfusion system used for preservation in step 402, such as system 100 described above in connection with FIG. 1. The sample contains nucleic acid. Step 406 involves isolating the nucleic acid from the sample. Step 408 involves performing an analysis of the isolated nucleic acid to assess at least one of the amount of nucleic acid in the perfusate, the molecular weight of the nucleic acid in the perfusate, or the fragment size distribution of the nucleic acid in the perfusate. Steps 404, 406, and 408 can be performed according to any of the implementations described above in connection with steps 202 / 302, 204 / 304, and 206 / 306 of Figures 2 and 3, respectively. Step 410 involves generating appropriate adjustments to make to 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, molecular weight, or fragment size distribution (or a subset thereof) of the nucleic acid). Step 412 involves adjusting the perfusion parameters of the machine perfusion to a second value based on the generated appropriate adjustments.

[0066] Steps 410 and 412 may be performed automatically by a controller, such as controller 120 described above in connection with FIG. 1. The controller may include machine-readable instructions for performing feedback control of perfusion parameters. Multiple perfusion parameters may be feedback-controlled simultaneously. The controller may use, but is not limited to, proportional control, proportional-integral control, or proportional-integral-derivative (PID) control. Feedback control may be performed in response to user input of the analysis results from step 408. Alternatively, machine perfusion may be performed on a system that allows user control of perfusion parameters, and adjustments of the perfusion parameters may be performed manually by a physician / user.

[0067] Perfusion parameters may include, but are not limited to, perfusion fluid flow rate, perfusion fluid temperature, perfusion fluid pH, perfusion fluid oxygen concentration, therapeutic agent (e.g., immunosuppressant or anti-inflammatory drug, stem cells) concentration, perfusion fluid component (e.g., salts, proteins, nutrients) concentration, or perfusion duration, etc. Appropriate adjustments may include recommendations for adjusting more than one perfusion parameter simultaneously or sequentially.

[0068] In some implementations, the method 400 further includes taking a post-adjustment sample of the perfusate and assessing whether further adjustment is required. The steps of adjusting and reassessing for further adjustment may be performed any number of times.

[0069] Further Technology Methods 200, 300, and / or 400 may further include, at any time after isolating nucleic acids from the perfusate sample, performing targeted genetic analysis of the isolated nucleic acids to identify one or more genetic features within the nucleic acids. Amplification and sequencing (e.g., high-throughput sequencing, microarray, nanopore sequencing) may be used to perform this targeted genetic analysis.

[0070] In some embodiments, the method includes isolating or purifying nucleic acids. There are several procedures for achieving this goal. In some embodiments, the sample may be centrifuged to separate the various layers. In some embodiments, the nucleic acids may be isolated using filtration. In some embodiments, the preparation of nucleic acids may involve amplification, separation, chromatographic purification, liquid-liquid separation, isolation, preferential enrichment, preferential amplification, target amplification, reverse transcription, or any combination thereof, or any of several other techniques described herein. In some embodiments for isolating DNA, RNase is used to degrade RNA. Alternatively, DNase may be used to degrade DNA and isolate RNA in the sample.

[0071] In some embodiments, universally tagged adapters are added to generate the library. Prior to ligation, the sample nucleic acids are blunt-ended and then a single adenosine base is added to the 3' end. In some implementations, the tagged adapters can be added using PCR. In some implementations, the tagged adapters can be added using ligation. Prior to ligation, the nucleic acids 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 increase ligation efficiency. In some embodiments, the library is amplified using universal primers. In one embodiment, the amplified library is fractionated by size separation or other methods. In some embodiments, PCR amplification is used to amplify the target loci. In some embodiments, the amplified nucleic acids are sequenced (e.g., 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 at a particular base when sequencing from one end of the amplified nucleic acid, there is less chance of a sequence error at 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).

[0072] In some embodiments, whole genome amplification (WGA) is used to amplify nucleic acid samples. Several methods can be used for WGA, including ligation-mediated PCR (LM-PCR), in which short DNA sequences called adapters are ligated to blunt ends of DNA. These adapters contain universal amplification sequences that are used to amplify DNA by PCR. In another embodiment, WGA is performed using degenerate oligonucleotide primer PCR (DOP-PCR), in which random primers that also contain universal amplification sequences are used in the first round of annealing and PCR. A second round of PCR is then used to further amplify the sequences using the universal primer sequences. In another embodiment, WGA is performed using multiple displacement amplification (MDA), which uses phi-29 polymerase, a highly processive and nonspecific enzyme that replicates nucleic acids and has been used in single-cell analysis. In some embodiments, WGA is not performed.

[0073] In some embodiments, selective amplification or enrichment is used to amplify or enrich target loci. In some embodiments, amplification and / or selective enrichment techniques may include PCR, such as ligation-mediated PCR, fragment capture by hybridization, molecular inversion probes, or other circularization 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 are used (Hung et al., J Clin Pathol 62:308-313, 2009, incorporated herein by reference in its entirety). In some embodiments, hybridization capture using hybrid capture probes is used to preferentially enrich nucleic acids. In some embodiments, methods for amplification or selective enrichment may involve using probes that, when properly hybridized to the target sequence, separate the 3' or 5' end of the nucleotide probe from the polymorphic site of the polymorphic allele by a small number of nucleotides. This separation reduces preferential amplification of one allele, known as allelic bias. This is an improvement over methods involving the use of probes in which the 3' or 5' end of a correctly hybridized probe is directly adjacent to or very close to the polymorphic site of an allele. In one embodiment, probes whose hybridizing region may or certainly contains the polymorphic site are excluded. Polymorphic sites at the hybridization site may cause unequal hybridization or completely inhibit hybridization at some alleles, resulting in preferential amplification of certain alleles. These embodiments are an improvement over other methods involving targeted amplification and / or selective enrichment in that they better preserve the original allele frequencies of a sample at each polymorphic locus, whether the sample is a pure genomic sample from a single individual or a mixture of individuals.

[0074] In some embodiments, a PCR technique called mini-PCR is used to generate very short amplicons (see U.S. Application No. 13 / 683,604, filed November 21, 2012; U.S. Publication No. 2013 / 0123120; U.S. Application No. 13 / 300,235, filed November 18, 2011; U.S. Publication No. 2012 / 0270212, filed November 18, 2011; and U.S. Application No. 61 / 994,791, filed May 16, 2014, each of which is incorporated herein by reference in its entirety). cfDNA is highly fragmented. In some cfDNA, fragment sizes are distributed in a roughly Gaussian pattern, with a mean of 160 bp, a standard deviation of 15 bp, a minimum size of approximately 100 bp, and a maximum size of approximately 220 bp. The polymorphic site of a particular target locus may occupy any position from the beginning to the end of various fragments derived from that locus. Because cfDNA fragments are short, the likelihood that both primer sites are present—that is, the likelihood that a fragment of length L contains both forward and reverse primer sites—is the ratio of the amplicon length to the length of the fragment. Under ideal conditions, assays with amplicons of 45, 50, 55, 60, 65, or 70 bp will successfully amplify 72%, 69%, 66%, 63%, 59%, or 56% of the available template fragment molecules, respectively. In certain implementations, cfDNA is amplified using primers that yield a maximum amplicon length of 85, 80, 75, or 70 bp, with a melting temperature of 50-65°C, and in certain preferred embodiments, 54-60.5°C. The amplicon length is the distance between the ends of the forward and reverse priming sites. Amplicon lengths shorter than those typically used may result in more efficient measurement of the desired polymorphic loci by requiring shorter sequence reads. In one embodiment, a substantial portion of the amplicons are less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp.

[0075] In some embodiments, amplification is performed using direct multiplex PCR, sequential PCR, nested PCR, duplex nested PCR, one-sided semi-nested PCR, fully nested PCR, one-sided fully nested PCR, one-sided nested PCR, hemi-nested PCR, hemi-nested PCR, triplex hemi-nested PCR, semi-nested PCR, one-sided semi-nested PCR, reverse semi-nested PCR, or one-sided PCR, as described in U.S. Application No. 13 / 683,604, filed November 21, 2012; U.S. Publication No. 2013 / 0123120; U.S. Application No. 13 / 300,235, filed November 18, 2011; U.S. Publication No. 2012 / 0270212; and U.S. Application No. 61 / 994,791, filed May 16, 2014, which are incorporated by reference in their entireties. If desired, either of these methods may be used for mini-PCR.

[0076] If necessary, the extension step of the PCR amplification can be time-limited to reduce amplification from fragments longer than 200, 300, 400, 500, or 1,000 nucleotides. This may improve test performance by enriching for fragmented or shorter nucleic acids (e.g., cfDNA from cells undergoing apoptosis or necrosis).

[0077] 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 generate a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to generate amplification products containing target amplicons. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplification products are primer dimers. In some embodiments, the primers are in solution (e.g., dissolved in a liquid phase rather than a solid phase). In some embodiments, the primers are in solution and not immobilized on a solid support. In some embodiments, the primers are not part of a microarray. In some embodiments, the primers do not comprise a molecular inversion probe (MIP).

[0078] In some embodiments, two or more (e.g., three or four) target amplicons (e.g., amplicons from the mini-PCR methods disclosed herein) are ligated together, and the ligated product is then sequenced. Combining multiple amplicons into a single ligation product improves the efficiency of the subsequent sequencing step. In some embodiments, the target amplicons are less than 150, 100, 90, 75, or 50 base pairs in length before they are ligated. Selective enrichment and / or amplification can involve tagging each individual molecule with a different tag, molecular barcode, tag for amplification, and / or tag for sequencing. In some embodiments, the amplification products are analyzed by sequencing (e.g., 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, for example, the World Wide Web at geniachip.com / technology, which is incorporated herein by reference in its entirety). In some embodiments, double-stranded 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 incorporated herein by reference in its entirety). This technique significantly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. Because the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors can be discounted as technical errors because mutations occur in only one strand. In some embodiments, the method involves tagging both strands of double-stranded DNA with random but complementary double-stranded nucleotide sequences, called double-stranded tags.Double-stranded tag sequences are incorporated into sequencing adapters by first introducing a single-stranded randomized nucleotide sequence into one adapter strand and then extending the opposite strand with DNA polymerase to generate a complementary double-stranded tag. After ligating the tagged adapter to sheared DNA, the individually labeled strands are PCR amplified from asymmetric primer sites on the adapter tails and subjected to paired-end sequencing. In some embodiments, a sample (e.g., a DNA sample) or nucleic acid preparation is divided into multiple fractions, such as different wells (e.g., wells of a WaferGen SmartChip). Dividing a sample or preparation into different fractions (e.g., at least 5, 10, 20, 50, 75, 100, 150, 200, or 300 fractions) can increase the sensitivity of the analysis by ensuring that the proportion of molecules carrying the mutation is higher in some wells than in the overall sample. In some embodiments, each fraction contains less than 500, 400, 200, 100, 50, 20, 10, 5, 2, or 1 nucleic acid molecule. In some embodiments, the molecules in each fraction are sequenced separately. In some embodiments, the same barcode (such as a random or non-human sequence) is added to all molecules in the same fraction (e.g., by amplification with barcode-containing primers or barcode ligation), and different barcodes are added to molecules in different fractions. The barcoded molecules can be pooled and sequenced together. In some embodiments, the molecules are amplified, such as using nested PCR, before being pooled and sequenced. In some embodiments, one forward primer and two reverse primers, or two forward primers and one reverse primer, are used.

[0079] In some embodiments, the mutations (SNVs or CNVs, etc.) present in 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 cfDNA sample) or nucleic acid preparation are measured (or can be measured).In some embodiments, the mutations (SNVs or CNVs, etc.) 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 cfDNA sample from a blood sample, a perfusate sample or a flush sample) or nucleic acid preparation are measured (or can be measured).In some embodiments, the mutations (SNVs or CNVs, etc.) present in only one original nucleic acid molecule (before amplification) in a sample (such as a cfDNA sample from a blood sample, a perfusate sample or a flush sample) or nucleic acid preparation are measured (or can be measured).

[0080] For example, if the detection limit for a mutation (such as a single nucleotide variation (SNV)) is 0.1%, 0.01% mutations can be measured by dividing a sample into multiple fractions, such as 100 wells. The majority of wells contain no copies of the mutation. For the few wells that do contain a mutation, the mutation accounts for a much higher proportion of the reads. In one example, there are 20,000 initial copies of DNA from the target locus, and two of these copies contain the SNV of interest. When a sample or preparation is divided into 100 wells, 98 wells will have SNVs, and two wells will have 0.5% SNVs. The nucleic acid from each well is barcoded, amplified, pooled with the nucleic acid from the other wells, and sequenced. The SNV-free wells can be used to measure the background amplification / sequencing error rate and assess whether the signal from the outlier wells exceeds background levels of noise.

[0081] In some embodiments, the amplification products are measured using an array, particularly an array such as a microarray with probes for one or more chromosomes of interest (e.g., chromosomes 13, 18, 21, X, Y, or any combination thereof). It will be appreciated that SNP detection microarrays can be used, such as, for example, Illumina's (San Diego, CA) GoldenGate, DASL, Infinium, or CytoSNP-12 genotyping assays, or SNP detection microarray products from Affymetrix, such as OncoScan microarrays. In some embodiments, phased genetic data for one or both biological parents of the embryo or fetus is used to improve the accuracy of the analysis of array data from a single cell.

[0082] In some embodiments involving sequencing, read depth is the number of sequencing reads that map to a particular locus. Read depth may be normalized across the total number of reads. In some embodiments relating to the read depth of a sample or preparation, read depth is the average read depth across the target locus. In some embodiments relating to the read depth of a locus, read depth is the number of reads measured by the sequencer that map to that locus. Generally, the deeper the read depth of a locus, the closer the allele ratio at that locus tends to be to the allele ratio in the original sample or preparation of nucleic acid. Read depth can be expressed in a variety of different ways, including, but not limited to, percentages or ratios. Thus, for example, on a highly parallel DNA sequencer, such as the Illumina HISEQ, which generates 1 million clonal sequences, if a locus is sequenced 3,000 times, the read depth at that locus will be 3,000 reads. The percentage of reads at that locus is 3,000 divided by 1 million total reads, or 0.3% of the total reads.

[0083] In some embodiments, allele data is obtained, and the allele data includes quantitative measurements that indicate the copy number of a particular allele at a polymorphic locus. In some embodiments, the allele data includes quantitative measurements that are indicative of the copy number of each observed allele at a polymorphic locus. Quantitative measurements may be obtained for all possible alleles at a polymorphic locus of interest. For example, any of the methods discussed in the preceding paragraph for assessing alleles at SNP or SNV loci, such as DNA sequencing, e.g., microarrays, qPCR, RNA sequencing, high-throughput DNA sequencing, etc., can be used to generate quantitative measurements of the copy number of a particular allele at a polymorphic locus. This quantitative measurement is referred to herein as allele frequency data or measured genetic allele data. Methods that use allele data are sometimes referred to as quantitative allele methods, in contrast to quantitative methods that use only quantitative data from non-polymorphic loci or quantitative data from polymorphic loci but do not consider the identity of the allele. If the allele data is measured using high-throughput sequencing, the allele data may include the number of reads for each allele that maps to the locus of interest.

[0084] In some embodiments, non-allelic data are obtained and include quantitative measurements(s) indicating the copy number of a particular locus. A locus may be polymorphic or non-polymorphic. In some embodiments when a locus is non-polymorphic, the non-allelic data does not include information about the relative or absolute amounts of individual alleles that may be present at the locus. Methods that use only non-allelic data (i.e., quantitative data from non-polymorphic alleles, or quantitative data from polymorphic alleles but without considering the allelic identity of each fragment) are referred to as quantitative methods. Quantitative measurements may be obtained for all possible alleles at a polymorphic locus of interest, and a single value is associated with the measurements for all alleles at the locus as a whole. Non-allelic data for a polymorphic locus may be obtained by summing the quantitative alleles for each allele at the locus. If allelic data is measured using high-throughput sequencing, the non-allelic data may include the number of reads mapped to the locus of interest. The sequencing measurements can indicate the relative and / or absolute number of each allele present at that locus, and the non-allelic data includes the sum of reads mapped to that locus, regardless of allelic identity. In some embodiments, the same set of sequencing measurements can be used to obtain both allelic and non-allelic data. In some embodiments, the allelic data is used as part of a method for assessing copy number in a subject's chromosome, and the generated non-allelic data can be used as part of a separate method for assessing copy number in a subject's chromosome. In some embodiments, the two methods are statistically orthogonal and are combined to provide a more accurate assessment of copy number in a subject's chromosome.

[0085] In some embodiments, obtaining genetic data includes (i) obtaining nucleic acid sequence information by laboratory techniques, such as using an automated high-throughput sequencer, or (ii) obtaining information previously obtained by laboratory techniques that is transmitted electronically, e.g., by computer via the internet or by electronic transfer from a sequencing device.

[0086] Additional exemplary sample preparation, amplification, and quantification methods are described in U.S. Application No. 13 / 683,604, filed November 21, 2012 (U.S. Publication No. 2013 / 0123120, and U.S. Publication No. 61 / 994,791, filed May 16, 2014, which are incorporated by reference in their entireties). These methods can be used to analyze the samples or preparations disclosed herein.

[0087] Improved PCR amplification methods have also been developed that minimize or prevent interference from amplifying adjacent or neighboring target loci within 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 adjacent or neighboring target loci, which are faster and less expensive than methods that require dividing adjacent target loci into different reaction volumes so that the target loci can be amplified separately and avoid interference.

[0088] In some embodiments, amplification of target loci is performed using a polymerase (e.g., a DNA polymerase or a reverse transcriptase) with low 5' to 3' exonuclease activity and / or strand displacement activity. In some embodiments, low levels of 5' to 3' exonuclease reduce or prevent degradation of adjacent primers (e.g., primers that are not extended or primers that have one or more nucleotides added during primer extension). In some embodiments, low levels of strand displacement activity reduce or prevent displacement of adjacent primers (e.g., primers that are not extended or primers that have one or more nucleotides added during primer extension). In some embodiments, target loci that are adjacent to each other (e.g., no bases between the target loci) or nearby (e.g., loci within 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base apart) 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.

[0089] 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 amplified products are target amplicons. In various embodiments, the amount of amplified products that are target amplicons is between 50 and 99.5%, inclusive, e.g., between 60 and 99%, 70 and 98%, 80 and 98%, 90 and 99.5%, or 95 and 99.5%. 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., at least 5-, 10-, 20-, 30-, 50-, or 100-fold compared to the amount before amplification), e.g., by co-amplification in one reaction volume. In various embodiments, the amount of target loci that is amplified (e.g., at least 5-, 10-, 20-, 30-, 50-, or 100-fold compared to the amount before amplification) is 50-99.5%, e.g., 60-99%, 70-98%, 80-99%, 90-99.5%, 95-99.9%, or 98-99.99%, inclusive. In some embodiments, fewer non-target amplicons are produced, e.g., fewer amplicons are formed from the forward primer from the first primer pair and the reverse primer from the second primer pair. Such unwanted non-target amplicons can be produced using conventional amplification methods, for example, when the reverse primer from the first primer pair and / or the forward primer from the second primer pair are degraded and / or displaced.

[0090] In some embodiments, these methods allow for the use of longer extension times because the polymerase bound to the primer being extended is less likely to degrade and / or displace nearby primers (such as the next downstream primer) due to the polymerase's reduced 5' to 3' exonuclease and / or strand displacement activity. In various embodiments, reaction conditions (such as extension time and temperature) are used such that the polymerase extension rate results in the number of nucleotides added to the primer being extended that is 80, 90, 95, 100, 110, 120, 130, 140, 150, 175, or 200% or more of the number of nucleotides between the 3' end of the primer binding site and the 5' end of the next downstream primer binding site on the same strand.

[0091] In some embodiments, a DNA polymerase is used to generate a DNA amplicon using DNA as a template. In some embodiments, an RNA polymerase is used to generate an RNA amplicon using DNA as a template. In some embodiments, a reverse transcriptase is used to generate a cDNA amplicon using RNA as a template.

[0092] In some embodiments, the low level of 5' to 3' exonuclease activity of a polymerase is comparable to the same amount of Thermus aquaticus polymerase ("Taq" polymerase, a commonly used DNA polymerase derived from a thermophilic bacterium) under the same conditions (PDB 1BGX, EC2.7.7.7, 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, incorporated herein by reference in its entirety). 95:12562-12567, 1998). In some embodiments, the low level strand displacement activity of the 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 under the same conditions.

[0093] 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 incorporated by reference in its entirety). PHUSION DNA polymerase is a Pyrococcus-like enzyme fused to a processivity-enhancing domain. PHUSION DNA polymerase has 5' to 3' polymerase activity and 5' to 3' exonuclease activity, generating blunt-ended products. PHUSION DNA polymerase lacks 5' to 3' exonuclease activity and strand displacement activity.

[0094] In some embodiments, the polymerase is Q5® DNA polymerase, such as Q5® High-Fidelity DNA polymerase (M0491S, New England BioLabs, Inc.) or Q5® Hot Start High-Fidelity DNA polymerase (M0493S, New England BioLabs, Inc.). Q5® High-Fidelity DNA polymerase is a high-fidelity thermostable DNA polymerase with 3' to 5' exonuclease activity fused to an Sso7d domain, which enhances processivity. Q5® High-Fidelity DNA polymerase lacks 5' to 3' exonuclease activity and strand displacement activity.

[0095] In some embodiments, the polymerase is T4 DNA polymerase (M0203S, New England BioLabs, Inc.; Tabor and Struh. (1989). "DNA-Dependent DNA Polymerases," In Ausebel et al. (Ed.), 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, each of which is incorporated by reference in its entirety). T4 DNA polymerase catalyzes DNA synthesis in the 5' to 3' direction and requires the presence of a template and primer. This enzyme possesses a 3' to 5' exonuclease activity that is much more active than that found in DNA polymerase I. T4 DNA polymerase lacks 5' to 3' exonuclease and strand displacement activities.

[0096] 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 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, JP et al. (2006). Nucleic Acids Res., 34, 1102-1111, each of which is incorporated by reference in its entirety). Sulfolobus DNA Polymerase IV lacks 5' to 3' exonuclease activity and strand displacement activity.

[0097] In some embodiments, when a primer binds to a region containing a SNP, the primer may bind to and amplify different alleles with different efficiencies, or may bind to and amplify only one allele. For heterozygous subjects, one allele may not be amplified by the primer. In some embodiments, a primer is designed for each allele. For example, when there are two alleles (e.g., a biallelic SNP), two primers may be used to bind to the same position of the target locus (e.g., a forward primer for binding to the "A" allele and a forward primer for binding to the "B" allele). Methods such as, but not limited to, the dbSNP database can be used to evaluate the location of known SNPs, such as SNP hotspots with high heterozygosity rates.

[0098] 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 (e.g., amplification of target loci within fragmented nucleic acids), the length of the target loci is 50-100 nucleotides, inclusive, e.g., 60-80 nucleotides, or 60-75 nucleotides. In some embodiments (e.g., amplification of multiple target loci of exons or entire genes), the length of the target amplicon is 100-500 nucleotides, inclusive, e.g., 150-450 nucleotides, 200-400 nucleotides, 200-300 nucleotides, or 300-400 nucleotides.

[0099] In some embodiments, multiple target loci are simultaneously amplified using a primer pair comprising a forward primer and a reverse primer for each target locus to be amplified in the reaction volume. In some embodiments, a first PCR is performed using a single primer for each target locus, and then a second PCR is performed using a set of primer pairs for each target locus. For example, the first PCR may be performed using a single primer for each target locus (e.g., using a forward primer for each target locus) so that all primers bind to the same strand. This allows PCR to amplify in a linear manner, reducing or eliminating amplification bias between amplicons due to sequence or length differences. In some embodiments, the amplicons are then amplified using a forward primer and a reverse primer for each target locus.

[0100] If necessary, multiplex PCR may be performed using primers with a low likelihood of primer dimer formation. In particular, highly multiplexed PCR can often result in the production of a very high percentage of product nucleic acids due to unproductive side reactions such as primer dimer formation. In one embodiment, specific primers that are most likely to cause unproductive side reactions may be removed from the primer library to obtain a primer library with a high percentage of amplified nucleic acids that map to the genome. The step of removing problematic primers, i.e., primers that are particularly likely to stabilize dimers, unexpectedly allows for extremely high PCR multiplex levels for subsequent sequencing analysis.

[0101] There are several methods for selecting primers for libraries that minimize the amount of non-mapping primer-dimers or other primer artifacts. Empirical data indicates that a small number of "bad" primers are responsible for 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 sequence data of nucleic acids amplified by target amplification and remove the most frequently occurring primer dimers to obtain a primer library that is significantly less likely to produce side-product nucleic acids that do not map to the genome. There are also programs that can calculate the binding energy of various primer combinations. Removing those with the highest binding energies can also result in a primer library that is significantly less likely to produce side-product nucleic acids that do not map to the genome.

[0102] In some embodiments for selecting primers, an initial library of candidate primers is created by designing one or more primers or primer pairs for candidate target loci. The set of candidate target loci (e.g., SNPs) can be selected based on information about desired parameters of the target loci, such as the frequency of the SNP or the heterozygosity rate of the SNP within the target population. In one embodiment, PCR primers can be designed using the Primer3 program (libprimer3 release 2.2.3, available on the World Wide Web at primer3.sourceforge.net, incorporated herein by reference in its entirety). If desired, 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 in a specific size range, and / or have other parameter characteristics. Starting with multiple primers or primer pairs per candidate target locus increases the likelihood that primers or primer pairs will remain in the library for most or all of the target loci. In one embodiment, the selection criteria can require that at least one primer pair per target gene remain in the library. In this way, when using the final primer library, most or all of the target loci will be amplified.This is desirable for applications such as screening for deletions or duplications at multiple positions in the genome, or screening for multiple sequences (e.g., polymorphisms or other mutations) associated with a disease or an increased risk of a disease.If a primer pair from the library produces a target amplicon that overlaps with the target amplicon produced by another primer pair, one of the primer pairs can be removed from the library to prevent interference.

[0103] In some embodiments, an "undesirability score" (higher scores indicate less desirable) is calculated (e.g., computationally) for most or all of the possible combinations of two primers from a library of candidate primers. In various embodiments, an undesirability score is calculated for at least 80, 90, 95, 98, 99, or 99.5% of the possible combinations of candidate primers in the library. Each undesirability score is based, at least in part, on the likelihood of dimer formation 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 a sequence (e.g., a polymorphism) at the target locus, the disease penetrance associated with a sequence (e.g., a polymorphism) at the target locus, 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 a recombination hotspot. In some embodiments, the specificity of a candidate primer for a target locus includes the likelihood that the candidate primer will misprime by binding to and amplifying a locus other than the target locus it is designed to amplify. In some embodiments, one or more or all mispriming candidate primers are removed from the library. In some embodiments, to increase the number of candidate primers to select from, candidate primers that may misprime are not removed from the library. When multiple factors are considered, the undesirability score may be calculated based on a weighted average of various parameters. Parameters may be assigned different weights based on their importance to the particular application for which the primer is used. In some embodiments, the primer with the highest undesirability score is removed from the library. If the removed primer is a member of a primer pair that hybridizes to one target locus, the other member of that primer pair may be removed from the library. The process of removing primers may be repeated as desired.In some embodiments, the selection method is performed until the undesirability scores for the candidate primer combinations remaining in the library are all equal to or below a minimum threshold, hi some embodiments, the selection method is performed until the number of candidate primers remaining in the library is reduced to a desired number.

[0104] In various embodiments, after the undesirability scores are calculated, candidate primers that are part of the maximum number of two candidate primer combinations whose undesirability scores exceed 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 one target locus, the other member of that primer pair may be removed from the library. The process of removing primers may be repeated as desired. In some embodiments, this selection method is performed until the undesirability scores of the candidate primer combinations remaining in the library are all equal to or below the first minimum threshold. If the number of candidate primers remaining in the library is higher than desired, the number of primers may be reduced by reducing the first minimum threshold to a lower second minimum threshold and repeating the process of removing primers. If the number of candidate primers remaining in the library is less 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, this selection method is performed until the undesirability scores for the candidate primer combinations remaining in the library are all equal to or less than the second minimum threshold, or until the number of candidate primers remaining in the library has been reduced to the desired number.

[0105] If desired, primer pairs that produce target amplicons that overlap with target amplicons produced by another primer pair can be separated into separate amplification reactions. For applications in which it is desirable to analyze all of the candidate target loci (instead of omitting them from analysis due to overlapping target amplicons), multiple PCR amplification reactions may be desirable.

[0106] These selection methods minimize the number of candidate primers that need to be removed from the library to achieve the desired reduction in primer dimers. By removing fewer candidate primers from the library, more (or all) of the target loci can be amplified using the resulting primer library.

[0107] Multiplexing a large number of primers places significant constraints on the assays that can be included. Assays that unintentionally interact will result in spurious amplification products. The size constraints of mini-PCR can pose further constraints. In one embodiment, starting with a very large number of potential SNP targets (from approximately 500 to over 1 million), one can attempt to design primers to amplify each SNP. If primers can be designed, one can attempt to identify primer pairs that are likely to form spurious products by evaluating the likelihood of spurious primer duplex formation between all possible primer pairs using published thermodynamic parameters for duplex formation. Primer interactions can be ranked by a scoring function associated with this interaction, and primers with the worst interaction scores are eliminated until the desired number of primers is met. If potentially heterozygous SNPs are most useful, the list of assays can also be ranked, and the assays that are most compatible with heterozygosity can be selected. Experiments have shown that primers with high interaction scores are most likely to form primer dimers. While it is not possible to eliminate all spurious interactions when multiplexing, it is essential to eliminate the primers or primer pairs with the highest in silico interaction scores, as they can dominate the entire reaction and severely limit amplification from the intended target. This procedure was performed to create multiplex primer sets consisting of up to, in some cases, more than 10,000 primers. Compared to the 10% of reactions in which the worst primers were not removed, this procedure offers considerable improvements, enabling amplification of 80%, 90%, 95%, 98%, and even 99% of target products as measured by sequencing of all PCR products. When combined with a partial semi-nested approach, as described previously, more than 90% and even 95% of amplicons can be mapped to the target sequence.

[0108] It should be noted that there are other methods for assessing which PCR probes are likely to form dimers. In one embodiment, simply analyzing a pool of nucleic acids amplified using a non-optimized primer set may identify problematic primers. For example, analysis can be performed using sequencing, and the most abundant dimers can be identified as those most likely to form dimers and removed. In one embodiment, the primer design method may be used in combination with the mini-PCR method described herein.

[0109] The use of a tag on a primer can reduce amplification and sequencing of primer-dimer products. In some embodiments, a primer includes an internal region that forms a loop structure containing a tab. In certain embodiments, a primer includes a 5' region specific to a target locus, an internal region that is not specific to the target locus and forms a loop structure, and a 3' region specific to the target locus. In some embodiments, the loop region can be located between two binding regions, where the two binding regions are designed to bind to consecutive or adjacent regions of the template nucleic acid. In various embodiments, the 3' region is at least 7 nucleotides in length. In some embodiments, the 3' region is 7 to 20 nucleotides in length, e.g., 7 to 15 nucleotides, or 7 to 10 nucleotides in length, inclusive. In various embodiments, a primer includes a 5' region that is not specific to a target locus (e.g., a tag or universal primer binding site), followed by a region specific to the target locus, an internal region that is not specific to the target locus and forms a loop structure, and a 3' region specific to the target locus. Using tag primers, the required target-specific sequence can be shortened to less than 20, 15, 12, or even 10 base pairs. This can occur by chance or can be incorporated into primer design if the target sequence is fragmented within the primer binding site. The advantages of this method include increasing the number of assays that can be designed for a given maximum amplicon length and reducing the "uninformative" sequencing of primer sequences. It can also be used in conjunction with internal tagging.

[0110] In one embodiment, the relative amount of non-productive products in multiplex target PCR amplification can be reduced by increasing the annealing temperature. When amplifying a library using the same tag as the target-specific primers, the annealing temperature can be higher compared to genomic DNA because the tag contributes to primer binding. In some embodiments, reduced primer concentrations are used, optionally with longer annealing times. In some embodiments, annealing times can be longer than 3 minutes, 5 minutes, 8 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 60 minutes, 120 minutes, 240 minutes, 480 minutes, or even 960 minutes. In certain exemplary embodiments, longer annealing times are used with lower primer concentrations. In various embodiments, longer than normal extension times are used, such as longer than 3, 5, 8, 10, or 15 minutes. In some embodiments, the primer concentration is as low as 50 nM, 20 nM, 10 nM, 5 nM, 1 nM, or even less than 1 nM. This surprisingly results in stable performance for highly multiplexed reactions, such as 1,000-plex, 2,000-plex, 5,000-plex, 10,000-plex, 20,000-plex, 50,000-plex, and even 100,000-plex reactions. In one embodiment, amplification uses 1, 2, 3, 4, or 5 cycles with long annealing times, followed by PCR cycles with tagged primers and more conventional annealing times.

[0111] To select target positions, one may start with a pool of candidate primer pair designs, create a thermodynamic model of potentially deleterious interactions between the primer pairs, and then use the model to eliminate designs that are incompatible with other designs in the pool.

[0112] In one embodiment, the system or method features a method for 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 polymorphisms or mutations measured). In some embodiments, the method includes ranking loci (e.g., ranking from highest to lowest) by the frequency or recurrence of polymorphisms or mutations (e.g., single-nucleotide mutations, insertions, or deletions, or any of the other mutations described herein) at each locus among subjects with the disease or disorder. In some embodiments, PCR primers are designed for some or all of the loci. During selection of PCR primers for a library of primers, primers for loci with a higher frequency or recurrence (higher-ranked loci) are preferred over loci with a 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. If desired, primers that are incompatible with other designs in the library (such as primers for highly ranked loci) may be included in a different PCR library / pool. In some embodiments, multiple libraries / pools (e.g., 2, 3, 4, 5, or more) are used in separate PCR reactions, allowing amplification of all (or most) of the loci represented by all libraries / pools. In some embodiments, this method is continued until enough primers are included in one or more libraries / pools so that the primers collectively capture the desired disease burden for the disease or disorder (e.g., by measuring at least 80, 85, 90, 95, or 99% of the disease burden).

[0113] In some implementations, the systems or methods described herein use a library of primers, such as primers selected from a library of candidate primers using any of the methods described herein. In some embodiments, the library includes primers that simultaneously hybridize to (or are capable of simultaneously hybridizing to) 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) 100-500, 500-1,000, 1,000-2,000, 2,000-5,000, 5,000-7,500, 7,500-10,000, 10,000-20,000, 20,000-25,000, 25,000-30,000, 30,000-40,000, 40,000-50,000, 50,000-75,000, or 75,000-100,000 different target loci, inclusive, in a single reaction volume. In various embodiments, the library includes primers that simultaneously amplify (or are capable of simultaneously amplifying) 1,000 to 100,000 different target loci in a single reaction volume, e.g., 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, inclusive. In some embodiments, the library contains primers that co-amplify (or are capable of co-amplifying) target loci in one reaction volume such that less than 60, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.5% of the amplification products are primer dimers.In various embodiments, the amount of amplification product that is primer dimer is 0.5-60%, inclusive, e.g., 0.1-40%, 0.1-20%, 0.25-20%, 0.25-10%, 0.5-20%, 0.5-10%, 1-20%, or 1-10%. In some embodiments, the primers co-amplify (or are capable of co-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 product is target amplicon. In various embodiments, the amount of amplification product that is target amplicon is 50-99.5%, inclusive, e.g., 60-99%, 70-98%, 80-98%, 90-99.5%, or 95-99.5%. In some embodiments, the primers simultaneously amplify (or are capable of simultaneously amplifying) target loci within a single reaction volume such that 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-fold compared to the amount before amplification). In various embodiments, the amount of target loci that is amplified (e.g., amplified at least 5-, 10-, 20-, 30-, 50-, or 100-fold compared to the amount before amplification) is between 50 and 99.5%, e.g., between 60 and 99%, 70 and 98%, 80 and 99%, 90 and 99.5%, 95 and 99.9%, or 98 and 99.99%, inclusive. In some embodiments, the library of primers comprises 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 primer pair comprises a forward test primer and a reverse test primer, and where each test primer pair hybridizes to a 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 that each hybridize to a different target locus, where the individual primers are not part of a primer pair.

[0114] In various embodiments, the concentration of each primer is less than 100, 75, 50, 25, 20, 10, 5, 2, or 1 nM, or less than 500, 100, 10, or 1 uM. In various embodiments, the concentration of each primer is between 1 uM and 100 nM, inclusive, e.g., between 1 uM and 1 nM, 1 to 75 nM, 2 to 50 nM, or 5 to 50 nM. In some embodiments, the GC content of the primers is between 30 and 80%, inclusive, e.g., between 40 and 70%, or 50 and 60%. In some embodiments, the GC content of the primers is less than 30, 20, 10, or 5%. In some embodiments, the GC content of the primers is between 5 and 30%, inclusive, e.g., between 5 and 20%, or 5 and 10%. In some embodiments, the melting temperature (Tm) of the test primer is between 40 and 80°C, inclusive, e.g., 50 and 70°C, 55 and 65°C, or 57 and 60.5°C. In some embodiments, the Tm is calculated with the Primer3 program (libprimer3 release 2.2.3) using the built-in SantaLucia parameters (available on the world wide web at primer3.sourceforge.net). In some embodiments, the melting temperature range of the primer is less than 15, 10, 5, 3, or 1°C. In some embodiments, the melting temperature range of the primer is between 1 and 15°C, inclusive, e.g., 1 and 10°C, 1 and 5°C, or 1 and 3°C. In some embodiments, the length of the primer is between 15 and 100 nucleotides, inclusive, e.g., 15 and 75 nucleotides, 15 and 40 nucleotides, 17 and 35 nucleotides, 18 and 30 nucleotides, or 20 and 65 nucleotides. In some embodiments, the primer length ranges from less than 50, 40, 30, 20, 10, or 5 nucleotides. In some embodiments, the primer length ranges from 5 to 50 nucleotides, inclusive, such as 5 to 40 nucleotides, 5 to 20 nucleotides, or 5 to 10 nucleotides. In some embodiments, the target amplicon length ranges from 50 to 100 nucleotides, inclusive, such as 60 to 80 nucleotides, or 60 to 75 nucleotides.In some embodiments, the range of lengths of the target amplicons is less than 50, 25, 15, 10, or 5 nucleotides. In some embodiments, the range of lengths of the target amplicons is 5 to 50 nucleotides, inclusive, e.g., 5 to 25 nucleotides, 5 to 15 nucleotides, or 5 to 10 nucleotides. In some embodiments, the library does not include a microarray. In some embodiments, the library includes a microarray.

[0115] In some embodiments, some (e.g., at least 80, 90, or 95%) or all of the adapters or primers contain one or more linkages between adjacent nucleotides other than naturally occurring phosphodiester bonds. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. In some embodiments, some (e.g., at least 80, 90, or 95%) or all of the adapters or primers contain a phosphorothioate (e.g., monothiophosphate) between the last 3' nucleotide and the penultimate 3' nucleotide. In some embodiments, some (e.g., at least 80, 90, or 95%) or all of the adapters or primers contain a phosphorothioate (e.g., monothiophosphate) within the last 2, 3, 4, or 5 nucleotides at the 3' end. In some embodiments, some (e.g., at least 80, 90, or 95%) or all of the adapters or primers contain a phosphorothioate (e.g., monothiophosphate) within at least 1, 2, 3, 4, or 5 nucleotides of the last 10 nucleotides at the 3' end. In some embodiments, such primers are less likely to be cleaved or degraded. In some embodiments, the primer does not include an enzyme cleavage site (such as a protease cleavage site).

[0116] Additional exemplary multiplex PCR methods and libraries are described in U.S. Application No. 13 / 683,604, filed November 21, 2012 (U.S. Publication No. 2013 / 0123120), and U.S. Application No. 61 / 994,791, filed May 16, 2014, each of which is incorporated by reference in its entirety. These methods and libraries can be used to analyze any of the samples or preparations disclosed herein and for use in any of the disclosed methods.

[0117] Example 1 Figures 5A through 9B show the results of a study performed on perfusate samples collected from donated kidneys. A cohort of seven donors donated kidneys, four of whom donated one kidney each and three of whom donated two kidneys each. The kidney samples are shown in Table 1. Four 10 mL aliquots were collected from 10 perfusions of these kidneys. Each aliquot was centrifuged, and the cell pellet was separated from the remainder of the perfusate. Cellular DNA was extracted from the cell pellet using the QIAAMP® DNA Micro Kit, and cfDNA was extracted from the perfusate using the "NICE chemistry" protocol. Extracted DNA was quantified using an Invitrogen QUBIT® Fluorimeter (using the dsDNA BR Assay Kit), and fragment size was assessed using an Agilent 2100 BIOANALYZER® System (using the High Sensitivity DNA Assay and DNA-1000 Assay). [Table 2]

[0118] Figures 5A and 5B show electropherograms of cell-free and cellular components of DNA from each kidney perfusate sample. The horizontal axis represents fragment size in base pairs (bp), and the vertical axis represents the relative levels of each fragment size based on fluorescence units (FU). In Figure 5A, the lower marker (LM) is shown as a sharp peak at 15 bp, and the upper marker (UM) is shown as a sharp peak at 1500 bp. In Figure 5B, the LM is shown as a sharp peak at 35 bp, and the UM is shown as a sharp peak at 10,380 bp. LM and UM are internal markers added during processing to ensure proper sizing of the sample and do not represent the levels of sample DNA.

[0119] As shown in Figure 5A, cfDNA is present in the perfusate sample as several small peaks of approximately 200 bp and 400 bp, as well as fragments of various sizes represented by higher molecular weights. This distribution of smaller cfDNA fragments (e.g., less than approximately 200 bp) may represent DNA that has been fragmented around nucleosomes and released from cells during apoptosis, as these peaks have similar heights and are evenly spaced across the spectrum, corresponding to the size of nucleosomes. In Figure 5B, cellular DNA is present almost exclusively as high molecular weight fragments (e.g., greater than 200 bp).

[0120] Figures 6A, 6B, and 6C show electropherograms of specific samples with different size profiles. Figure 6A shows the cfDNA size profile of a sample assessed to have the highest DNA concentration among all samples. In this particular sample, only high-molecular-weight fragments were present at detectable levels, while low-molecular-weight (nucleosomal) fragments were undetectable. The corresponding cellular DNA profile is shown in Figure 6B, which also shows only high-molecular-weight fragments present at detectable levels. Figure 6C shows the cfDNA electropherogram of a specific sample, AHGF262_L, which prominently displays a 166-bp fragment peak, indicating the presence of significant nucleosomal DNA.

[0121] The presence of nucleosome peaks in a sample can be used to assess the possible source of cfDNA, which may correlate with the health of the donor's organ / tissue. For example, the samples in Figure 6A and Figure 6B may flag mechanical organ / tissue damage due to surgery or immune cell release, but the cfDNA lacks nucleosome fragmentation (e.g., a hallmark of cell necrosis), while the sample in Figure 6C with fragment sizes of approximately 160-170 bp may be due to programmed cell death via apoptosis, which may persist after transplantation, while the DNA in the other representative samples may be the result of necrosis.

[0122] In Figure 7, the yield of cellular DNA in μg / L is plotted against the yield of cfDNA in μg / L for each sample. Although the amount of cellular DNA was only approximately 1.3% of the amount of cfDNA on average for each sample, there is a linear correlation between the relative yields of each DNA fraction. Therefore, the amount of cellular DNA or cfDNA can potentially be used to estimate the amount 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.

[0123] In Figure 8, the percent cfDNA yield relative to kidney weight is graphed for each sample. The average kidney weight is reported to be 130 g. The two elevated bars are from samples taken from two kidneys of donor AHF3305, whose electropherograms are shown in Figures 6A and 6B.

[0124] In Figures 9A and 9B, cfDNA and cellular DNA yields (μg / L, respectively) are plotted against perfusion time in hours (the length of time each kidney was perfused before perfusate samples were taken). In Figure 9A, there is a strong linear correlation between cfDNA yield and perfusion time. The linear correlation has a slope of 124, a y-intercept of 1470, and an R-squared value of 0.9. In Figure 9B, there is a linear correlation between cellular DNA yield and perfusion time. The slope is 1.5, the y-intercept is 13.1, and an R-squared value of 0.48. Notably, the sample with the longest perfusion time is the same sample with the highest cfDNA yield in Figure 8, suggesting that prolonged perfusion may have caused kidney damage.

[0125] The foregoing is merely illustrative of the principles of the present disclosure, and the device may be practiced in other than the described embodiments, which are presented for purposes of illustration and not limitation. It will be understood that while the device disclosed herein is shown for use in machine perfusion of a donor organ or tissue, it is also applicable to the perfusion of other biological entities.

[0126] Variations and modifications will occur to those skilled in the art after considering this disclosure. The disclosed features may be implemented in any combination and subcombination (including multiple subsidiary combinations and subcombinations) with one or more other features described herein. The various features described or illustrated above, including any components thereof, may be combined or integrated in other systems. Furthermore, certain features may be omitted or not implemented.

[0127] The described systems and methods can be performed by a physician or automatically on a machine perfusion system and / or a DNA analysis platform. The perfusion system and / or DNA analysis platform may include a data processing device. The systems and methods described herein may be performed remotely on another data processing device. The other data processing device may be directly or indirectly connected to the system / platform via a cloud application. The system / platform may communicate with the other data processing device in real time (or near real time).

[0128] In general, embodiments of the subject matter and functional operations described herein can be implemented in digital electronic circuitry, including the structures disclosed herein and their structural equivalents, or in computer software, firmware, or hardware, or one or more combinations thereof. Embodiments of the subject matter described herein 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 controlling 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 one or more combinations thereof. The term "data processing apparatus" encompasses all apparatuses, devices, and machines that process data, including, by way of example, a programmable processor, a computer, or multiple processors or computers. Apparatus includes, in addition to hardware, code that establishes the execution environment for the computer program in question, such as code constituting processor firmware, a protocol stack, a database management system, an operating system, or one or more combinations thereof. Propagated signals are artificially generated signals, such as machine-generated electrical, optical, or electromagnetic signals generated to encode information for transmission to an appropriate receiving apparatus.

[0129] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, and can be deployed in any form, either as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program may correspond to a file in a file system. A 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 document), in a single file dedicated to the program, or in multiple coordinated files (such as files storing one or more modules, subprograms, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers located at one site or distributed across multiple sites and interconnected by a communications network.

[0130] The processes and logic flows described herein may be performed by one or more programmable processors executing one or more computer programs that perform functions that operate on input data and generate output. The processes and logic flows may also be performed by, and apparatus may be implemented as, special purpose logic circuitry, such as an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit).

[0131] Processors suitable for the execution of a computer program include, by way of example, both general-purpose and special-purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor receives instructions and data from a read-only memory, a random-access memory, or both. The essential elements of a computer are a processor for performing instructions and one or more memory devices for storing instructions and data. Typically, a computer also includes one or more mass storage devices, such as magnetic, magneto-optical, or optical disks, for storing data, or is operatively coupled to receive data from them, transfer data to them, or both. However, a computer need not have such devices.

[0132] Examples of changes, substitutions, and alterations are ascertainable by one skilled in the art and can be made without departing from the scope of the information disclosed herein. All references cited herein are incorporated by reference in their entirety and made a part of this application.

Claims

1. A method for preparing non-natural nucleic acid samples from perfusion fluid or flush useful for evaluating the transplantation results of donor organs or donor tissues, Obtaining a sample of the perfusion fluid or flush from a donor organ or donor tissue that has been perfused with the perfusion fluid or prepared with a flush, wherein the perfusion fluid or flush contains nucleic acids. To isolate all nucleic acids from the aforementioned sample, To measure at least one of the following: the amount of nucleic acid in the perfusion solution or flash, the molecular weight of the nucleic acid in the perfusion solution or flash, or the fragment size distribution of the nucleic acid in the perfusion solution or flash, the isolated nucleic acid to be assayed, Methods that include...

2. The method according to claim 1, wherein the amount of nucleic acid is the total amount of DNA in the perfusion solution or flush.

3. The method according to claim 1, wherein the amount of nucleic acid is the amount of high molecular weight DNA in the perfusion solution or flash.

4. The method according to claim 3, wherein the high molecular weight DNA includes a DNA fragment exceeding 200 base pairs.

5. The method according to claim 1, wherein the amount of nucleic acid is the amount of low molecular weight DNA in the perfusion solution or flash.

6. The method according to claim 5, wherein the low molecular weight DNA includes a DNA fragment of 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 solution or flash.

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

9. The method of claim 8, further comprising determining that the cell-free DNA originates from apoptosis if the cell-free DNA has a molecular weight that matches that of nucleosomal DNA.

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

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

12. The method according to claim 11, further comprising evaluating the gene expression characteristics corresponding to the isolated RNA to determine the cellular origin of the RNA.

13. The method according to claim 12, wherein, if the gene expression characteristics are related to immune function, the cellular origin of the RNA is evaluated to be an immune cell.

14. The method according to claim 11, wherein isolating the RNA includes degrading a vesicle containing the RNA.

15. The method according to claim 1, further comprising normalizing the amount of nucleic acid with respect to perfusion time.

16. The method according to claim 1, further comprising normalizing the amount of nucleic acid with respect to at least one of the size, weight, volume, or surface area of ​​the donor organ or donor tissue.

17. The method according to claim 1, further comprising normalizing the amount of DNA with respect to the perfusion flow rate of the perfusion solution or the amount of flash of the flash.

18. The method according to claim 1, further comprising normalizing the amount of nucleic acid with respect to the temperature of the perfusion solution or flash.

19. The method according to claim 1, further comprising evaluating the fragmentation pattern of the nucleic acid.

20. The method according to claim 19, further comprising classifying the isolated nucleic acids, based on the fragmentation pattern, into nucleic acids derived from the random degradation of ruptured cells in the perfusion fluid or flush, and nucleic acids derived from apoptosis of cells in the donor organ or donor tissue.

21. Obtain one or more additional samples of the perfusion fluid or flush taken from a different point in time than the aforementioned sample, To quantify the amount of nucleic acids in the perfusion fluid or flush of the one or more additional samples, Based on the aforementioned sample and the one or more additional samples, the amount of nucleic acid in the perfusion fluid or flush is tracked over time, The method according to claim 1, further comprising:

22. The method according to claim 1, wherein the measurement comprises at least one of electrophoresis, mass spectrometry, fluorescence measurement, qPCR, or droplet PCR.

23. The method according to claim 1, comprising assaying the prepared isolated nucleic acid to measure at least one of the following: the amount of nucleic acid in the perfusion or flush, the molecular weight of the nucleic acid in the perfusion or flush, or the fragment size distribution of the nucleic acid in the perfusion or flush.

24. A method for evaluating the results of transplantation, A nucleic acid preparation is prepared according to the method of claim 1, The results of transplantation of the donor organ or donor tissue are evaluated based on the amount of nucleic acid, Methods that include...

25. The method according to claim 24, wherein the nucleic acid is cellular DNA from donor-derived cells, the predicted result is a good or bad prognosis predicted based on the cellular DNA, and the cellular DNA exhibits an immune response.

26. The method according to claim 24, wherein the nucleic acid is cell-free DNA, the prediction result is a good or bad prognosis predicted based on the cell-free DNA, and the cell-free DNA indicates damage to the donor organ or donor tissue.

27. The method according to claim 24, wherein the prediction result is a predicted poor prognosis that is a decisive factor in not using the organ for transplantation.

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

29. The method according to claim 24, wherein the prediction result is predicted by performing a quality assessment of the donor organ or donor tissue based on the amount of nucleic acid.

30. The method according to claim 29, wherein the quality assessment indicates at least one of the characteristics related to transplantation outcomes, such as the presence of graft function delay, the duration of graft function delay, the rate of early dysfunction, or organ function at various time points after transplantation.

31. A method for evaluating the results of transplantation, A nucleic acid preparation is prepared according to the method of claim 1, The results of the transplantation are evaluated based on at least a portion of the fragment size distribution of the nucleic acid, Methods that include...

32. The method according to claim 31, wherein the prediction result is predicted at least in part on risk calls generated by an algorithm that uses the fragment size distribution or a subset of the fragment size distribution as algorithmic input.

33. The method according to claim 24 or 31, wherein the predicted result is a rejection reaction to the transplant or a non-rejection reaction to the transplant.

34. The method according to claim 24 or 31, wherein the prediction result includes the type of rejection reaction to the transplant.

35. The method according to claim 24 or 31, wherein the prediction result includes the time of rejection of the transplant.

36. The method according to claim 24 or 31, further comprising recommending, based on the prediction results, that perfusion of the organ or tissue be stopped, that transplantation be postponed, or that transplantation be delayed.

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

38. The method according to claim 1, further comprising performing target gene analysis on the isolated nucleic acid to identify the genetic characteristics in the nucleic acid.

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

40. The method according to any one of claims 38 and 39, further comprising evaluating changes in homeostasis or cellular processes based on the aforementioned target gene analysis.

41. A method for preparing non-natural nucleic acid preparations from perfusion fluid or flush useful for evaluating the transplantation results of donor organs or donor tissues, Obtaining a sample of the perfusion fluid or flush from a donor organ or donor tissue that has been perfused with the perfusion fluid or prepared with a flush, wherein the perfusion fluid or flush contains nucleic acids. To isolate nucleic acids from the aforementioned sample, The isolated nucleic acid is prepared so that it can be assayed for at least one of the types of nucleic acids in the sample or the sequences of the nucleic acids in the sample. Methods that include...

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

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

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

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

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

47. A method for feedback-controlled mechanical perfusion of a donor organ or donor tissue, Perfusing the donor organ or donor tissue in a perfusion chamber holding the donor organ or donor tissue at a first value of the perfusion parameter, The method according to claim 1 is applied to the donor organ or donor tissue, To generate appropriate adjustments to the perfusion parameters based at least partially on the measurement of the isolated nucleic acids, Based on the appropriate adjustments generated, the perfusion parameters are adjusted to a second value. Methods that include...

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 reduced to the minimum threshold perfusion flow rate necessary to maintain the transplant in a state in which the perfusion flow rate is 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, immunosuppressants, nutrients, or red blood cells.

53. The method according to any one of claims 47 to 50, wherein the perfusion parameter is the temperature or pH of the perfusion solution.

54. After preparing the perfusion solution, take a sample, To evaluate whether additional adjustments are needed, The method according to any one of claims 47 to 50, further comprising:

55. The method according to any one of claims 1, 24, 31, 41, and 47, wherein the donor organ is a kidney, lung, heart, liver, gallbladder, pancreas, or intestine.

56. The method according to any one of claims 1, 24, 31, 41, and 47, 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, 24, 31, 41, and 47, 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, 24, 31, 41, and 47, wherein the donor tissue is blood or platelets.

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