Method for determining DNA or cell chimerism in individual

Abstract

To provide a method for determining DNA or cell chimerism in an individual.SOLUTION: Provided is a method for detecting nucleic acid derived from a first subject and / or nucleic acid derived from a second subject in a biological sample derived from the first subject, including the steps of: obtaining polymorphism information for each of 25 or more types of polymorphisms for each nucleic acid contained in the biological sample, the polymorphisms including Indel and / or Microhaplotypes; and determining an abundance ratio of nucleic acid derived from the first subject and / or an abundance ratio of nucleic acid derived from the second subject contained in the biological sample from the polymorphism information.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to methods for determining DNA or cellular chimerism in an individual. [Background technology]

[0002] Hematopoietic stem cell transplantation is an effective treatment for hematopoietic tumors (Non-Patent Document 1). Minimal residual disease (MRD) significantly affects the prognosis of patients (Non-Patent Document 2). Known methods for analyzing chimerism include short tandem repeat (STR) analysis (Non-Patent Document 3) and XY-FISH (Non-Patent Document 4). The sensitivity of these methods is about 1-5%, and the accuracy rate is about 10 6 STR may not be used to determine chimerism. XY-FISH can only determine chimerism between males and females.

[0003] Patent Document 1 discloses a method for detecting or quantifying the donor source of a nucleic acid from a mixture of nucleic acids or cells derived from different clones or individuals. The method of Patent Document 1 requires error correction using error-corrected sequence reads.

[0004] Patent Document 2 discloses the detection of single nucleotide polymorphisms and copy number variants.

[0005] Non-Patent Document 5 discloses the generation of amplicon panels.

[0006] Patent Document 3 discloses a method for assessing the risk of developing colorectal cancer, which detects the presence of at least 28 single nucleotide polymorphisms.

[0007] Patent Document 4 discloses the detection of more than 72 single nucleotide polymorphisms associated with breast cancer. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2019 / 222560 [Patent Document 2] WO2020 / 168239 [Patent Document 3] WO2017 / 127893 [Patent Document 4] WO2016 / 049694 [Non-patent literature]

[0009] [Non-Patent Document 1] Heidrich et al., Ann. Oncl., 2017, 28(11):2793~2798 [Non-Patent Document 2] Kim et al., Blood, 2018, 132, 1604~1613 [Non-Patent Document 3] Tiede et al., Bone Marrow Transplant., 1999, 23(10), 1055~1060 [Non-Patent Document 4] Wedi et al., Reprod. Fertil. Dev. 2016, 29(5), 913~920 [Non-Patent Document 5] Lee et al., J. Clin. Med., 2019, 8, 2077 Summary of the Invention

[0010] The present disclosure provides methods for determining DNA or cellular chimerism in an individual.

[0011] According to the present disclosure, for example, the following inventions are provided. (1) A method for detecting a nucleic acid derived from a first subject and / or a nucleic acid derived from a second subject in a biological sample derived from a first subject, comprising: obtaining polymorphism information for each of 25 or more polymorphisms for each nucleic acid contained in the biological sample, wherein the polymorphisms include indels and / or microhaplotypes; determining the abundance ratio of nucleic acid derived from the first subject and / or the abundance ratio of nucleic acid derived from the second subject contained in the biological sample from the polymorphism information; Including, the step of obtaining the polymorphism information includes obtaining a plurality of types of amplified fragments, the plurality of amplified fragments including regions containing all of the polymorphisms; obtaining the amplified fragments includes linking a common adapter sequence to one end of the region including each of the polymorphisms in each of the nucleic acids, and obtaining each amplified fragment including the region using a primer for the adapter sequence and a primer for a sequence specific to the amplified fragment located at the other end of the region; the first subject and the second subject have an allogeneic relationship; The method includes determining to which of the two types of sequences the sequence of a region containing each polymorphism in each nucleic acid is more similar, thereby determining the abundance ratio of nucleic acids derived from a first subject and / or the abundance ratio of nucleic acids derived from a second subject. (2) The method according to (1) above, wherein the first subject has had the experience of receiving a transplant of cells derived from the second subject. (3) The method described in (2) above, wherein the first subject has undergone transplantation of hematopoietic stem cells derived from the second subject in the treatment of a hematopoietic tumor, and the determining step includes determining the proportion of nucleic acid derived from the first subject. (4) The method described in (2) above, wherein the first subject has undergone transplantation of mesenchymal stem cells derived from the second subject, and the determining step includes determining the proportion of nucleic acid derived from the second subject. (5) The method according to any one of (1) to (4) above, wherein the 25 or more types of polymorphisms include 30 or more types of indels. (6) The method according to any one of (1) to (5) above, wherein the nucleic acid is cell-free DNA. (7) The method according to any one of (1) to (5) above, wherein the nucleic acid is intracellular DNA or RNA. (8) A method according to any one of (1) to (7) above, wherein the determining step comprises determining the abundance ratio of one, two or three nucleic acids selected from the group consisting of a nucleic acid derived from a first subject, a nucleic acid derived from a second subject, and a nucleic acid derived from a third subject, contained in a biological sample, from polymorphism information, and the first subject, the second subject, and the third subject each have an allogeneic relationship with the other subjects. (9) The method according to (8) above, wherein the first subject has undergone transplantation of cells derived from the second subject and transplantation of cells derived from the third subject. (10) The method described in (3) above, wherein the nucleic acid is cell-free DNA, and the presence of cell-free DNA derived from the first subject and / or an increase in the concentration of the cell-free DNA indicates a possibility of graft-versus-host disease (GVHD). (11) The method according to any one of (1) to (10) above, wherein the step of obtaining polymorphism information includes obtaining polymorphism information on each of 30 or more types of polymorphisms. (12) The method according to any one of (1) to (11) above, wherein the step of obtaining polymorphism information includes obtaining polymorphism information on each of 34 or more types of polymorphisms. (13) The method according to (4) above, which is performed on each of biological samples derived from a first subject and derived from two or more different tissues, and determines the abundance ratio of nucleic acid derived from a second subject for each of the two or more different tissues. (31) The method according to any one of (1) to (13) above, which does not include an error correction step. (32) The method according to (31) above, wherein the error correction step comprises linking an adapter (error correction sequence read) unique to the nucleic acid molecule to each nucleic acid molecule. (51) Any of the methods described above, wherein the steps from the start of obtaining information on polymorphisms from an obtained biological sample to determining the proportion of polymorphisms are carried out within two weeks. (52) Any of the methods described above, wherein the steps from the start of obtaining information on polymorphisms from an obtained biological sample to determining the abundance ratio are carried out within 2 days. (53) Any of the methods described above, wherein the steps from initiation of obtaining information on polymorphisms from an obtained biological sample to determination of the abundance ratio are carried out within one day. (71) Any of the above methods, wherein the subject is a human and the first subject and the second subject are HLA-compatible. (72) Any of the above methods, wherein the subject is a human and the first subject and the second subject are HLA half-matched. (73) Any of the above methods, wherein the subject is a human and the first subject and the second subject are HLA1 antigen incompatible. (74) Any of the above methods, wherein the subject is a human and the first subject and the second subject are HLA2 antigen incompatible. (75) Any of the above methods, wherein the subject is a human and the first subject and the third subject are HLA-compatible. (76) Any of the above methods, wherein the subject is a human and the first subject and the third subject are HLA half-matched. (77) Any of the above methods, wherein the subject is a human and the first subject and the third subject are HLA1 antigen incompatible. (78) Any of the above methods, wherein the subject is a human and the first subject and the third subject are HLA2 antigen incompatible. (79) The method according to any one of (71) to (74) above, wherein the subject is a human, and the first subject and the third subject are HLA-compatible. (80) The method according to any one of (71) to (74) above, wherein the subject is a human, and the first subject and the third subject are HLA half-matched. (81) The method according to any one of (71) to (74) above, wherein the subject is a human, and the first subject and the third subject are HLA1 antigen incompatible. (82) The method according to any one of (71) to (74) above, wherein the subject is a human, and the first subject and the third subject are HLA2 antigen incompatible. (91) The method according to any of the above, wherein the indel and / or microhaplotype is selected to satisfy one or more or all of the following conditions selected from the group consisting of: the surroundings of the polymorphism (e.g., about 20 bp upstream and downstream including the polymorphism) are unique sequences in the human genome; there are no sequences in the human genome with a homology of 90% or more, 80% or more, 70% or more, 60% or more, or 50% or more; the surroundings of the polymorphism (e.g., about 20 bp upstream and downstream including the polymorphism) contain no mismatches, contain only 1 to 3 mismatches, contain only 2 or less, or contain only 1 or less mismatches; the indel and / or microhaplotype is selected from the above 500, top 400, top 300, top 200, top 150, or top 100 mismatches with high Wootton-Federhen values; the indel and / or microhaplotype is not present in a repetitive sequence; and the indel and / or microhaplotype does not share a specific sequence pattern with other sequences. (92) The method according to any of the above, wherein the indel and / or microhaplotype is selected to satisfy one or more or all of the following: the surroundings of the polymorphism (e.g., about 20 bp upstream and downstream of the polymorphism) are unique sequences in the human genome; there are no sequences in the human genome with a homology of 90% or more, 80% or more, 70% or more, 60% or more, or 50% or more; the surroundings of the polymorphism (e.g., about 20 bp upstream and downstream of the polymorphism) contain no mismatches or contain only one or less mismatches; the indel and / or microhaplotype is selected from the top 200, top 150, or top 100 sequences with high Wootton-Federhen values; the indel and / or microhaplotype is not present in a repetitive sequence; and the indel and / or microhaplotype does not share a specific sequence pattern with other sequences. (93) The method according to any of the above, wherein the indel and / or microhaplotype is selected to satisfy one or more or all of the following conditions selected from the group consisting of: the surroundings of the polymorphism (e.g., about 20 bp upstream and downstream of the polymorphism) are unique sequences in the human genome; there are no sequences in the human genome with which the homology is 50% or more; the surroundings of the polymorphism (e.g., about 20 bp upstream and downstream of the polymorphism) contain no mismatches or contain only one or less mismatches; the indel and / or microhaplotype is selected from the top 100 sequences with the highest Wootton-Federhen values; the indel and / or microhaplotype is not present in a repetitive sequence; and the indel and / or microhaplotype does not share a specific sequence pattern with other sequences. [Brief description of the drawings]

[0012] [Figure 1] A scheme for determining polymorphisms from sequence reads is shown. [Diagram 2] The results of 75 indel polymorphisms for 175 cases are shown. [Diagram 3] 4 shows the detection accuracy of polymorphisms at the indicated dilution concentrations. [Figure 4] The results of chimerism measurement for case 1 are shown. [Figure 5A] 1 shows the chimerism analysis scheme for case 2. [Figure 5B] The results of measuring the chimerism of B cells, NK cells, and T cells in PBMNCs from case 2 are shown. [Figure 5C] 1 shows the results of verification of the chimerism measurement results of case 2 based on the STAT3 mutation rate. [Figure 6A] 1 shows the two cell transplantation schemes from different subjects in case 3. [Figure 6B] The results of measuring the amount of cell-free DNA (cfDNA) in the blood and chimerism in whole blood in case 3 are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As used herein, a "subject" may be a mammal, particularly including rodents such as rats and mice, non-rodent mammals such as horses, goats, cows, pigs, sheep, dogs, and cats, and primates such as humans. A subject may also be referred to as an individual. Humans include, but are not limited to, Negroids (black race group), Caucasoids (white race group), Mongoloids (yellow race group), and Australoids (black-brown race group). The first subject, second subject, and third subject are different individuals from each other, and are allogeneic or xenogeneic to each other, particularly allogeneic.

[0014] As used herein, "nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA can be genomic DNA, intracellular DNA or cell-free DNA. Cell-free DNA is extracellular DNA in body fluids (e.g., in blood).

[0015] As used herein, a "biological sample" is a sample derived from an organism. A biological sample may be obtained from a living organism. A biological sample may be a body fluid or tissue. Body fluids include, for example, saliva, tears, sweat, blood, lymphatic fluid, tissue fluid, body cavity fluid (e.g., pleural fluid, peritoneal fluid, pericardial fluid), cerebrospinal fluid, synovial fluid, aqueous humor, digestive fluid (pancreatic juice, gastric juice, bile, intestinal juice), urine, nasal mucus, semen, vaginal fluid, amniotic fluid, and milk. Body fluids contain nucleic acids. Tissues include, for example, epithelial tissues (e.g., protective epithelium, secretory epithelium, sensory epithelium, and absorptive epithelium), connective tissues (proper connective tissue, specialized connective tissue, and embryonic connective tissue), muscle tissue, and nerve tissue. Proper connective tissues include loose connective tissues (tissues that hold organs and epithelia), dense connective tissues (e.g., ligaments and tendons), adipose tissue, and reticular tissue (e.g., cartilage lattices that support lymphatic organs). Special connective tissues include bone and cartilage. Embryonic connective tissues include mesenchymal and gelatinous tissues. Muscle tissues include skeletal and visceral muscles (e.g., striated, smooth, and cardiac).

[0016] In this specification, a "polymorphism" is an individual difference in a nucleic acid sequence on genomic DNA possessed by a subject. Typically, when a difference is observed in a certain nucleic acid sequence in a population at a rate of 1% or more, the difference is called a polymorphism. Typically, a polymorphism does not have a fatal effect on a phenotype. A polymorphism is scattered on genomic DNA. Therefore, a polymorphism is broadly divided into a genetically linked polymorphism and a genetically unlinked polymorphism. The polymorphism that occupies the largest proportion is called a reference sequence, and the others are sometimes called variants.

[0017] In this specification, "allogeneic" means that the organism is of the same species but originates from a different individual. Allogeneic is roughly divided into cases where the major histocompatibility complex (MHC; in humans, called human leukocyte antigen (HLA)) is completely matched and cases where it is not completely matched. In humans, HLA compatibility is called when all HLA-A, B, and DR, each of which has two alleles, are matched, and HLA half-match is called when any of paternally derived and maternal HLA-A, B, and DR are completely matched. When one HLA locus is mismatched and the rest are matched, it is called HLA1 antigen mismatch, and when two HLA loci are mismatched and the rest are matched, it is called HLA2 antigen mismatch. When hematopoietic stem cells from a donor with one or more HLA loci mismatched are transplanted into a recipient, the recipient may develop graft-versus-host disease (GVHD). Hematopoietic stem cells are present in bone marrow and umbilical cord. Hematopoietic stem cells have the ability to self-renew and differentiate into all blood cells. Hematopoietic stem cells reside primarily in the bone marrow and can be detected in the blood of an individual following administration of granulocyte colony stimulating factor (G-CSF) to said individual.

[0018] As used herein, "donor" refers to the individual providing the stem cells, and "recipient" refers to the individual receiving the transplant. The recipient is also called the host.

[0019] In this specification, "hematopoietic stem cell transplantation" means transplantation of hematopoietic stem cells. Hematopoietic stem cell transplantation can be performed to treat subjects with blood cancer or immunodeficiency. Hematopoietic stem cell transplantation is performed on subjects who have undergone pre-transplant treatment. Pre-transplant treatment can be performed to reduce tumor cells and suppress the subject's own immune cells. Pre-transplant treatment is broadly divided into myeloablative treatment and non-myeloablative treatment. Myeloablative treatment has a high antitumor effect, but has strong side effects and is limited to cases where certain criteria are met in terms of age and systemic symptoms. In pre-transplant treatment, for example, a large amount of anticancer drug is administered or whole-body radiation treatment is performed. Hematopoietic stem cells are administered to patients who have undergone pre-transplant treatment. Administration is usually performed by intravenous drip (infusion). Typically, antihistamines or steroids are administered to prevent fever and allergic reactions. Subjects who have undergone hematopoietic stem cell transplantation can receive G-CSF treatment. Engraftment is when the neutrophil count reaches 500 cells / μL or more and this state continues for 3 days or more. Blood cancer may recur after hematopoietic stem cell transplantation. Patients who have relapsed may receive chemotherapy or radiation therapy. Patients who have relapsed may receive a second hematopoietic stem cell transplant. Patients who have relapsed may receive donor lymphocyte infusion (DLI). DLI is indicated for patients who have undergone hematopoietic stem cell transplantation but have failed to engraft, relapsed, or developed a severe viral infection. Donor lymphocytes are obtained from the donor by whole blood collection or component blood collection.

[0020] As used herein, "peripheral blood stem cell transplantation" refers to transplantation of hematopoietic stem cells obtained from the blood of a subject who has received G-CSF administration. Peripheral blood stem cell transplantation is performed on a subject who has received pre-transplant treatment. Hematopoietic stem cells in the blood of a subject who has received G-CSF administration are typically collected from the subject's blood using a blood cell separator.

[0021] As used herein, "mesenchymal stem cells" refer to stem cells present in adults, and have the ability to differentiate into mesodermal tissues (e.g., bone, cartilage, blood vessels, and cardiomyocytes), ectodermal tissues (e.g., nerve cells and glial cells), and endodermal tissues (e.g., hepatocytes). Mesenchymal stem cells (MSCs) can be isolated from bone marrow, fat, and umbilical cord. Mesenchymal stem cells can be administered for vascular regeneration or for cardiac regeneration.

[0022] As used herein, "indel" refers to either or both of an insertion of a nucleic acid sequence into genomic DNA and a deletion of a nucleic acid sequence from genomic DNA. An indel is typically a small genetic mutation of up to 10,000 base pairs, and a small indel of up to 50 base pairs is sometimes called a microindel. It is believed that there are about 190 to 280 frameshift indels in humans. A short indel is an indel of 8 base pairs or less, preferably 6 base pairs or less, and particularly 4 base pairs or less. Indels can be present in the coding region, non-coding region, and gene translation region of a gene.

[0023] As used herein, a "microhaplotype" refers to a combination of multiple single nucleotide polymorphisms (SNPs), e.g., two, preferably three, and more preferably four or more SNPs, within a region of 200 base pairs or less (e.g., 100 base pairs or less, particularly 70 base pairs or less, and preferably 50 base pairs or less).

[0024] As used herein, "single nucleotide polymorphism" refers to a difference of one base found in a DNA sequence between individuals. Typically, a single nucleotide polymorphism is said to occur when the difference occurs at a frequency of 1% or more or 5% or more in a population. Single nucleotide polymorphisms can occur in coding regions, non-coding regions, and gene coding regions of genes.

[0025] In this specification, "chimerism" means the mixing ratio of cells derived from two or more different individuals. In particular, in hematopoietic stem cell transplantation or peripheral blood stem cell transplantation, it means the mixing ratio of donor-derived cells and recipient-derived cells in blood cells. Chimerism in an individual who has undergone two or more cell transplants means the mixing ratio of donor-derived cells and recipient-derived cells in each of the two transplants. In particular, when two cell transplants are performed, cells derived from the first donor, cells derived from the second donor, and cells derived from the recipient may be mixed. Chimerism may vary for each tissue or cell type of the recipient. For example, the chimerism of a recipient who has undergone mesenchymal stem cell (MSC) transplantation is thought to differ in the engraftment rate of MSCs for each tissue, and may show different chimerism for each tissue. Chimerism is the mixing ratio of cells, but it can also be determined as the ratio of donor-derived nucleic acid and recipient-derived nucleic acid in nucleic acid such as genomic DNA extracted from a cell group. Chimerism focusing on the mixing ratio of cells is referred to as cellular chimerism in this specification. Chimerism can be used to predict the recurrence of tumors such as leukemia after stem cell transplantation. Specifically, when recipient-derived cells are detected in the blood after hematopoietic stem cell transplantation, tumor recurrence is suspected. Chimerism can also be cell-free DNA chimerism. Cell-free DNA is extracellular DNA. Cell-free DNA (cfDNA) is thought to be mainly leaked from destroyed cells. Destruction can be caused by immune cells and cell death such as apoptosis. Analysis of cell-free DNA can be used to detect cancer, autoimmune diseases, graft rejection, graft-versus-host disease, and early fetal abnormalities in pregnant women. Blood cfDNA levels in healthy individuals can be 50 ng / mL or less. cfDNA can be collected from an individual's body fluids, but preferably from blood.

[0026] According to the present disclosure, a method is provided for detecting nucleic acid from a first subject and / or nucleic acid from a second subject in a biological sample from a first subject. This method can be used to determine chimerism. The first subject and the second subject can be a recipient and a donor, respectively. For example, in hematopoietic stem cell transplantation and peripheral blood stem cell transplantation, the first subject can be a recipient and the second subject can be a donor. Also, in cell transplantation, tissue transplantation, or organ transplantation, such as mesenchymal stem cell transplantation, the first subject can be a recipient and the second subject can be a donor.

[0027] Nucleic acids from the first subject and / or nucleic acids from the second subject may be detected for one or more cell types contained in the biological sample, and thus the methods of the disclosure may further comprise isolating, concentrating, enriching, or purifying said one or more cell types from the biological sample from the first subject.

[0028] According to the present disclosure, 1. A method for detecting a nucleic acid from a second subject in a biological sample from a first subject, comprising: obtaining polymorphism information regarding one or more polymorphisms (e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, or 75 or more polymorphisms, e.g., 34 or more and 75 or less polymorphisms) for each nucleic acid contained in the biological sample; determining the abundance ratio of nucleic acid derived from the first subject and / or the abundance ratio of nucleic acid derived from the second subject contained in the biological sample from the polymorphism information; The first subject and the second subject are different subjects, e.g., having an allogeneic relationship.

[0029] In a preferred embodiment, the polymorphism may be selected from those with an allele frequency of 1:9 to 1:1, those with a frequency of 1:8 to 1:1, those with a frequency of 1:7 to 1:1, those with a frequency of 1:6 to 1:1, those with a frequency of 1:5 to 1:1, those with a frequency of 1:4 to 1:1, those with a frequency of 1:3 to 1:1, those with a frequency of 1:2 to 1:1, and those with a frequency of 1:1. The closer the ratio of the reference sequence to the variant is to 1:1, the more preferable. In a preferred embodiment, the polymorphism may be selected from those with sufficient sequence complexity. For example, the polymorphism may be selected based on the index that the 20 bp interval surrounding it is unique on the human genome and that there are three or less mismatches. Furthermore, in a preferred embodiment, the polymorphism may be selected from those that are considered to be effective for the race to which the human individual belongs. When the ratio of the major allele to the minor allele in the relevant race is at least 50:50 to 90:10, the relevant polymorphism is considered to be valid.

[0030] In a preferred embodiment, the polymorphism comprises or consists of indels and / or microhaplotypes.

[0031] In a preferred embodiment, the polymorphism is selected based on the criterion that the surroundings (for example, about 20 bp upstream and downstream including the polymorphism) are unique sequences in the human genome. A unique sequence may mean that there is no sequence in the human genome with a homology of 90% or more, 80% or more, 70% or more, 60% or more, or 50% or more. In a preferred embodiment, the surroundings (for example, about 20 bp upstream and downstream including the polymorphism) of the polymorphism do not contain any mismatches, contain only 1 to 3 mismatches, contain only 2 or less mismatches, or contain only 1 or less mismatches. In a preferred embodiment, the polymorphisms can be selected in descending order of Wootton-Federhen value determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993). For example, the polymorphisms can be selected from the above 500, top 400, top 300, top 200, top 150, or top 100 Wootton-Federhen values. In some preferred embodiments, the polymorphism is not present in a repeat sequence. In some preferred embodiments, the polymorphism does not share a particular sequence pattern with other sequences. In some preferred embodiments, the polymorphism may be selected from those having some or all of the characteristics described in this paragraph. For example, in a preferred embodiment, the polymorphism may be selected to satisfy one or more or all of the following: the surrounding area (e.g., about 20 bp upstream and downstream including the polymorphism) is a unique sequence in the human genome; there are no sequences in the human genome with which it has 90% or more, 80% or more, 70% or more, 60% or more, or 50% or more homology; the surrounding area (e.g., about 20 bp upstream and downstream including the polymorphism) contains no mismatches, only 1 to 3 mismatches, only 2 or less, or only 1 or less; the polymorphism is selected from the above-mentioned 500, top 400, top 300, top 200, top 150, or top 100 mismatches with the highest Wootton-Federhen values; it is not present in a repetitive sequence; and it does not share a specific sequence pattern with other sequences. A polymorphism may be a significant polymorphism if the minor polymorphism in a population represents 5% or more, 10% or more, 15% or more, or 20%-50% of the population.

[0032] In one embodiment, the first subject has undergone transplantation of cells derived from the second subject (e.g., stem cells, particularly hematopoietic stem cells, blood stem cells such as peripheral blood stem cells, etc.). In another embodiment, the first subject has undergone transplantation of cells derived from the second subject (e.g., stem cells, particularly tissue stem cells such as mesenchymal stem cells, etc.). In another embodiment, the first subject has undergone transplantation of hematopoietic stem cells derived from the second subject in the treatment of hematopoietic tumors, and the determining step comprises determining the proportion of nucleic acid derived from the first subject. In another embodiment, the first subject has undergone transplantation of mesenchymal stem cells derived from the second subject, and the determining step comprises determining the proportion of nucleic acid derived from the second subject.

[0033] In some embodiments, the nucleic acid may be DNA or RNA. In some embodiments, the nucleic acid may be intracellular DNA or intracellular RNA. In some embodiments, the nucleic acid may be cell-free nucleic acid (specifically cell-free DNA or DNA or RNA contained in extracellular vesicles). In some embodiments, the nucleic acid may be contained in extracellular vesicles (e.g., apoptotic vesicles, exosomes, or microvesicles (MVs).

[0034] In the method of the present disclosure, the step of obtaining the polymorphism information includes obtaining a plurality of types of amplified fragments (preferably the plurality of amplified fragments include a region containing all of the polymorphisms), and obtaining the amplified fragments includes linking an adapter sequence common to the nucleic acids to one end of a region containing each polymorphism in each nucleic acid (particularly a fragment of a nucleic acid), and obtaining each amplified fragment containing the region using a primer for the adapter sequence and a primer for a sequence unique to the amplified fragment located at the other end of the region. In this embodiment, a nucleic acid fragment may be used as the nucleic acid. The nucleic acid fragment may be obtained, for example, by treating the nucleic acid with an enzyme that cleaves the nucleic acid, but is not particularly limited thereto. In addition, an adapter for amplification with a universal primer may be ligated to one end of the cleaved nucleic acid fragment, and an amplified fragment may be obtained with a primer for a sequence specific to the amplified fragment and a universal primer. In this way, the occurrence of errors may be reduced. By making the polymorphism an indel and / or a microhaplotype and amplifying it in the above process, the polymorphism analysis can be accelerated and the error occurrence rate during the analysis can be reduced.

[0035] In the method of the present disclosure, the step of obtaining the polymorphism information may not include a step of concentrating the nucleic acid fragments (i.e., pre-amplified fragments) containing the polymorphism or the amplified fragments. This is to prevent enrichment bias of the fragments due to the enrichment step. The enrichment may be performed, for example, using a surface (plate, beads, etc.) on which a nucleic acid having a sequence complementary to the nucleic acid fragments containing the polymorphism or the amplified fragments is immobilized. In the method of the present disclosure, the step of obtaining the polymorphism information may not include a step of removing amplification errors in the amplified fragments (error removal step). An example of the error removal step is disclosed, for example, in WO2013 / 142389. For example, this error removal step includes linking an adapter (error correction sequence read) specific to the nucleic acid molecule to each nucleic acid molecule. This error removal step may also include comparing the sequences of amplified nucleic acid fragments having the same adapter sequence to remove errors (those with a small number of reads (minor errors) among the bases that differ in the same adapter). In this case, the polymorphisms may be selected to include, preferably consist of, for example, indels and / or microhaplotypes, and more preferably consist of short indels and / or microhaplotypes.

[0036] In some embodiments, the amplified fragment has a length of 100bp or more, 200bp or more, 300bp or more, 400bp or more, 500bp or more, 600bp or more, 700bp or more, 800bp or more, 900bp or more, 1000bp or more, 15000bp or more, 2000bp or more, 25000bp or more, 3000bp or more. In some embodiments, the amplified fragment has a length of 3000bp or less, 2500bp or less, 2000bp or less, 1500bp or less, 1000bp or less, 900bp or less, 800bp or less, 700bp or less, 600bp or less, 500bp or less, 400bp or less, 300bp or less, or 200bp or less. In some embodiments, the amplified fragment can have a length of 200bp to 3000bp, 500bp to 2000bp, or 1000bp to 1500bp.

[0037] The method of the present disclosure comprises: The determining step determines to which of the two sequences the sequence of the region containing each polymorphism in each nucleic acid has a higher similarity, thereby determining the abundance ratio of the nucleic acid derived from the first subject and / or the abundance ratio of the nucleic acid derived from the second subject. This allows detection of proliferation of recipient-derived cells after hematopoietic stem cell transplantation (e.g., tumor recurrence) and detection of the engraftment rate of donor-derived cells in the recipient after stem cell transplantation.

[0038] In one embodiment, the engraftment of cells in a tissue is determined, for example, by performing a biological sample from a first subject derived from one tissue and determining the abundance ratio of nucleic acid derived from a second subject for said tissue. The engraftment of cells in a tissue is determined, for example, by performing a biological sample from a first subject derived from two or more different tissues and determining the abundance ratio of nucleic acid derived from a second subject for said two or more different tissues.

[0039] In the method of the present disclosure, the rate of isomerization is 1 in 10 molecules, 1 in 20 molecules, 1 in 30 molecules, 1 in 40 molecules, 1 in 50 molecules, 1 in 60 molecules, 1 in 70 molecules, 1 in 80 molecules, 1 in 90 molecules, 1 in 100 molecules, 1 in 200 molecules, 1 in 300 molecules, 1 in 400 molecules, 1 in 500 molecules, 1 in 600 molecules, 1 in 700 molecules, 1 in 800 molecules, 1 in 90 molecules, 1 in 100 molecules, 1 in 200 molecules, 1 in 300 molecules, 1 in 400 molecules, 1 in 500 molecules, 1 in 600 molecules, 1 in 700 molecules, 1 in 800 molecules, 1 in 900 molecules, 1 in 1 ... It may be possible to detect the abundance of nucleic acid derived from a first subject or the abundance of nucleic acid derived from a second subject, present at 1 in 00, 1 in 900, 1 in 1000, 1 in 2000, 1 in 3000, 1 in 4000, 1 in 5000, 1 in 6000, 1 in 7000, 1 in 8000, 1 in 9000, or 1 in 10,000.

[0040] In the method of the present disclosure, the time from the start of the process of obtaining polymorphism information of the obtained biological sample to the determination of the abundance ratio can be, for example, within 2 weeks, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 42 hours, within 36 hours, within 30 days, or within 1 day. The process can be simplified and may be kept within this time frame.

[0041] Whether to detect the cells (genome) of the first subject or the cells (genome) of the second subject may be determined depending on the purpose.

[0042] In one embodiment, the first subject has undergone a transplant of hematopoietic stem cells derived from the second subject in the treatment of a hematopoietic malignancy, and the determining step determines the proportion of nucleic acid derived from the first subject, in this embodiment, detection of or an increased proportion of nucleic acid or cells derived from the first subject may be indicative of minimal residual disease.

[0043] In one embodiment, the first subject has undergone transplantation of cells (e.g., mesenchymal stem cells) derived from a second subject, and the determining step determines the proportion of nucleic acid derived from the second subject. In this embodiment, detection of or an increase in the proportion of nucleic acid or cells derived from the second subject indicates engraftment of the cells (e.g., mesenchymal stem cells) in the tissue sample from which they were derived.

[0044] Whether cfDNA derived from a first subject or cfDNA derived from a second subject is detected can be determined depending on the purpose.

[0045] In one embodiment, the first subject has undergone transplantation of hematopoietic stem cells derived from the second subject in the treatment of hematopoietic tumors, and the step of determining determines the presence ratio of cfDNA derived from the first subject.The presence of cfDNA or an increase in the ratio of cfDNA may indicate the onset of graft-versus-host disease (GVHD).This is because immune cells derived from transplanted hematopoietic stem cells attack the cells of the first subject, destroy the attacked cells, and cfDNA is released from the destroyed cells into body fluids.

[0046] In some embodiments, the nucleic acid is intracellular nucleic acid (e.g., DNA and RNA. In some embodiments, the nucleic acid is free outside the cell. In some embodiments, the nucleic acid (e.g., DNA or RNA) is present in an extracellular vesicle (e.g., an exosome, apoptotic vesicle, or MV).

[0047] In one embodiment of the present disclosure, the determining step in the above invention includes determining the abundance ratio of one, two or three nucleic acids selected from the group consisting of a nucleic acid derived from a first subject, a nucleic acid derived from a second subject, and a nucleic acid derived from a third subject contained in a biological sample from polymorphism information. In this embodiment, preferably, the first subject, the second subject, and the third subject each have an allogeneic relationship with the other subject. In a preferred embodiment, the first subject has undergone a transplant of cells derived from the second subject and a transplant of cells derived from the third subject. In this embodiment, the method preferably includes determining the abundance ratio of each of the three nucleic acids, the nucleic acid derived from the first subject, the nucleic acid derived from the second subject, and the nucleic acid derived from the third subject. In this manner, the proportion of cells or nucleic acids derived from each subject in a mixture of cells or nucleic acids derived from three or more subjects can be determined. In one embodiment, the number of subjects can be four or more, and the method of the present disclosure can determine the proportion of cells or nucleic acids derived from each subject in a mixture of cells or nucleic acids derived from each of the four or more subjects. EXAMPLES

[0048] 1. Target Selection To ensure the validity of SNP target loci, we considered loci with 1-4 bp insertions or deletions (indels) whose allele frequency in the Japanese population was close to 0.5 according to the Tohoku Medical Megabank Organization (ToMMo) database. These candidate loci were narrowed down by ensuring that the 20 bp interval surrounding them was unique in the human genome and had no more than three mismatches. Loci with the highest Wootton-Federhen values, as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993), and those without repeats or patterns were manually inspected and selected as targets for indels. In addition, a set of microhaplotypes from the Allelic Frequency Database (Yale University) was added to the target list. Finally, 85 SNP loci were selected as targets.

[0049] 2. Sample Preparation To verify the detection of single nucleotide polymorphisms (SNPs), peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral venous blood (10 ml) of healthy subjects by Ficoll density gradient centrifugation and stored deep frozen until DNA extraction. NA12878 (Coriell Institute for Medical Research) genomic DNA was also used as a control. To quantify the texture of patients after transplantation, PBMNCs and plasma samples were sequentially isolated from peripheral venous blood before and after transplantation. Genomic DNA was isolated using the QIAmp DNA Micro Kit (Qiagen) and stored at 4°C until analysis.

[0050] Sequencing libraries targeting SNP loci were prepared using the QIAseq Targeted DNA Panel (Qiagen) according to the manufacturer's instructions. Genomic DNA (10-40 ng) was fragmented, end-repaired, and polyA-tailed. Adapter oligos containing a 12-base random sequence (i.e., unique molecular identifier; UMI) and a sample index were ligated to the 5' end of the prepared DNA fragments. Target enrichment was performed by 8 cycles of PCR using region-specific primers for each SNP locus and a universal primer complementary to the adapter. The enriched fragments were subjected to an additional 20 cycles of universal PCR reaction to add adapter sequences to complete the final library structure.

[0051] Prepared libraries were quantified and subjected to 150 bp paired-end sequencing on Illumina platforms (MiSeq, NextSeq 500, NextSeq 2000) using a custom sequencing primer for Read 1 (QIAseq A Read1 Primer I).

[0052] 3. Variant detection After error correction by base substitution and grouping by UMI, the resulting DNA sequences (reads) were aligned to the standard human genome with indels at the target locus. Because accidental indel errors were more likely to occur at the ends of reads, fragments with reads close to the target indel site were excluded from further analysis. Furthermore, because indel alignments may not be unique due to the influence of DNA sequences around the target SNPs, alignments with multiple indels nearby were recalibrated for re-presentation against the intended SNPs. The allele ratio at a site was also estimated from the number of matched read fragments and the number of read fragments with mutations (see Figure 1).

[0053] 4. Estimation of Mixture Ratio A set of SNPs was selected to pre-estimate the recipient / donor admixture ratio in transplant samples, taking into account allele and copy number information, if partially available. This initial estimate was refined by setting up a hierarchical model to estimate the allele state at selected loci. This general model was applied to samples with multiple contributions, corresponding to non-binary cases.

[0054] Example 1 Using the above method, 75 indel polymorphisms were determined, and 175 human subjects were examined based on the indel polymorphisms. As a result, an average of 4 to 5 indel differences were found between two individuals (see Figure 2). In addition, each of the 175 cases had a different set of indels, making it possible to distinguish between individuals (see Figure 2).

[0055] Example 2 DNA extracted from two individuals was mixed at 1:1 to 1:10. -4 The mixture ratio (chimerism) was estimated (see Figure 3). -4 The mixture ratio (chimerism) could be predicted well within the range (see Figure 3).

[0056] Example 3 (Case 1) A human case (female, 32 years old) was diagnosed with acute myeloid leukemia (ALL (IgH-EPOR)) and subsequently underwent allogeneic bone marrow transplantation (unrelate, DR1miss) with the administration of blinatumomab. In bone marrow transplantation, recipient-derived hematopoietic stem cells are destroyed and donor-derived bone marrow is transplanted, resulting in the construction of a donor-derived blood cell system in the recipient's body. The recipient genome polymorphisms and donor genome polymorphisms were determined by the above-mentioned method, and peripheral blood mononuclear cells were collected from the female recipient to determine the genome mixing ratio (chimerism). As a result, while the female recipient's genome was 100% before transplantation, the peripheral blood mononuclear cells collected 30 days after transplantation contained 1.98% of the recipient's genome, the peripheral blood mononuclear cells collected 70 days after transplantation contained 5.58% of the recipient's genome, and the peripheral blood mononuclear cells collected 196 days after transplantation contained 15.4% of the recipient's genome (see FIG. 4). This suggests that the female recipient's own white blood cells had increased in her body despite having undergone bone marrow transplantation. The chimerism determined by the method of this embodiment was close to the chimerism determined by the XY-FISH method.

[0057] Example 4 (Case 2) The human case (female, 35 years old) was a patient with hyper-IgE syndrome (HIES) with a mutation in STAT3. The female recipient underwent allogeneic peripheral blood stem cell transplantation (relate, full match). We determined the recipient polymorphism and donor polymorphism, and determined the chimerism of T cells, NK cells, and B cells in peripheral blood mononuclear cells collected from the female recipient 2,512 days after transplantation (see Figure 5A). The results showed that B cells contained 5.13% of the recipient genome, NK cells contained 1.01% of the recipient genome, and T cells contained 29.8% of the recipient genome (see Figure 5B). STAT3 in the transplanted peripheral blood stem cells and STAT3 in the recipient genome had different sequences due to the mutation. The STAT3 mutation ratios in the collected T cells, NK cells, and B cells were determined and compared with the chimerism based on the above polymorphisms. As shown in Figure 5C, the results showed high consistency with each other.

[0058] Example 5 (Case 3) The human case (22 years old, elastic) was a patient with acute myeloid leukemia (AML) with a mutation in U2AF1. The male recipient underwent allogeneic peripheral blood stem cell transplantation (relate, full match) and then further allogeneic mesenchymal stem cell transplantation (relate, haplo) (see Figure 6A). The recipient then developed grade III (S2L3G4) graft-versus-host disease (GVHD). Peripheral blood samples were obtained from the male recipient before and after MSC transplantation (Pre and Post), and chimerism was analyzed. The recipient-derived genomic polymorphisms, transplanted peripheral blood stem cell (MSC)-derived genomic polymorphisms, and mesenchymal stem cell-derived genomic polymorphisms were determined, and the chimerism of cell-free DNA (cfDNA) and whole blood in the obtained samples was determined. As a result, the recipient-derived genomic chimerism in cfDNA before MSC transplantation (Pre) was 2.63%. After MSC transplantation (Post), the recipient-derived genome in the cfDNA was 1.18%, and the MSC-derived genome in the cfDNA was 0.30% (see FIG. 6B). In contrast, the MSC-derived genome in the whole blood (WBC) was 0.15% (see FIG. 6B). The presence of cfDNA suggests that the cells have been destroyed. The male recipient had developed GVHD, and it is believed that the peripheral blood stem cell transplant damaged the recipient-derived cells, which is why the cfDNA was detected. The chimerism of the recipient genome-derived cfDNA decreased after MSC transplantation, suggesting that MSC transplantation alleviated the symptoms of GVHD. Thus, the method of this example can determine the chimerism of cells derived from two or more individuals in one individual. In addition, by focusing on cfDNA, the degree of cell damage (e.g., the severity of GVHD) can be detected. In addition, the method of this example can be useful for determining the presence or absence and the degree of engraftment of MSC in various tissues after MSC transplantation. The method of the present invention allows analysis to be completed within 1 to 2 days, and is considered to be advantageous for rapid analysis.

[0059] By analyzing 34 or more polymorphic sites, it is theoretically possible to cover the diversity of the entire population on Earth. In practice, chimerism analysis can be performed by examining 25 or more polymorphisms that cover 30 million variations, or 27 polymorphisms that cover 13,000 variations.

Claims

1. A method for detecting nucleic acids derived from a first target and / or from a second target in a biological sample derived from a first target, A step to obtain polymorphic information for each of 25 or more polymorphisms with respect to each nucleic acid contained in the biological sample, wherein the polymorphism includes indels and / or microhaplotypes. A step of determining the proportion of nucleic acids derived from a first target and / or a second target contained in a biological sample from the polymorphic information. Includes, The step of obtaining the polymorphism information includes obtaining multiple types of amplified fragments, wherein the multiple amplified fragments include regions containing all of the polymorphisms, and obtaining the amplified fragments includes ligating a common adapter sequence to one end of the region containing each polymorphism in each nucleic acid, and obtaining each amplified fragment containing the region using a primer for the adapter sequence and a primer for a sequence specific to the amplified fragment located at the other end of the region. The first object and the second object have a relationship of being of the same kind but different in origin. The method comprising the step of determining which of the two types of sequences the sequence of the region containing each polymorphism in each nucleic acid is more similar to, thereby determining the proportion of nucleic acids derived from the first target and / or the proportion of nucleic acids derived from the second target.

2. A method according to claim 1, wherein the first subject has experience receiving transplantation of cells derived from the second subject.

3. A method according to claim 2, wherein the first subject has experience receiving transplantation of hematopoietic stem cells derived from the second subject in the treatment of hematopoietic malignancies, and the determining step includes determining the proportion of nucleic acids derived from the first subject.

4. A method according to claim 2, wherein the first subject has experience receiving transplantation of mesenchymal stem cells derived from the second subject, and the determining step includes determining the proportion of nucleic acids derived from the second subject.

5. The method according to Claim 1, wherein the 25 or more polymorphs include 30 or more indels.

6. The method according to Claim 1, wherein the nucleic acid is cell-free DNA.

7. The method according to Claim 1, wherein the nucleic acid is intracellular DNA or RNA.

8. A method according to Claim 1, wherein the determining step includes determining the proportion of one, two, or three nucleic acids selected from the group consisting of nucleic acids derived from a first target, nucleic acids derived from a second target, and nucleic acids derived from a third target contained in a biological sample, wherein the first target, the second target, and the third target are each related to the other targets in a homogeneous, heterogeneous relationship.

9. A method according to claim 8, wherein the first subject has experience undergoing transplantation of cells derived from the second subject and transplantation of cells derived from the third subject.

10. A method according to claim 3, wherein the nucleic acid is cell-free DNA, and the presence of cell-free DNA derived from the first subject, and / or an increase in the concentration of such cell-free DNA, indicates the possibility of graft-versus-host disease (GVHD).

11. A method according to Claim 1, wherein the step of obtaining the polymorphism information includes obtaining polymorphism information relating to each of 30 or more types of polymorphisms.

12. A method according to Claim 1, wherein the step of obtaining the polymorphism information includes obtaining polymorphism information relating to each of the 34 or more types of polymorphisms.

13. A method according to claim 4, comprising performing the procedure on each biological sample derived from a first subject derived from two or more different tissues, for determining the proportion of nucleic acids derived from a second subject in each of the two or more different tissues.

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