Probe set for multi-sample KIR gene typing using the capture method and typing method using the same

Abstract

We will develop a probe set that can process multiple samples simultaneously using sequence capture methods and comprehensively capture genes in the KIR region. [Solution] A probe set for KIR gene typing is provided, each consisting of oligonucleotides having a specific base sequence. Furthermore, a method for KIR gene typing using the probe set is provided.

Description

Technical Field

[0001] The present invention relates to a novel probe set suitable for use in multi-sample KIR gene typing using the sequence capture method. More specifically, it relates to a probe set that can comprehensively and efficiently capture polymorphic KIR genes and enable simultaneous typing of multiple samples.

Background Art

[0002] The natural killer cell immunoglobulin-like receptor (KIR) gene family belongs to a plurality of receptor families that encode important proteins found on the surface of natural killer (NK) cells.

[0003] The KIR gene family includes 16 genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DP1, KIR3DS1 and KIR3DP1). The KIR gene cluster is located within a 100 - 200 kb region of the leukocyte receptor complex (LRC) on chromosome 19 (19q13.4). The number of copies and types of inherited KIR genes vary among human individuals, and KIR genotypes can vary greatly between individuals and within ethnic groups (Patent Document 1).

[0004] The KIR gene is expressed on the surface of NK cells and recognizes HLA molecules, regulating their function. Therefore, it is thought to play a crucial role in the human innate immune response and post-transplant rejection. However, the KIR gene has been difficult to analyze due to its high degree of diversity. This difficulty stems from the challenges of designing target sequences based on intraregional similarity, the extremely high number of genetic variations within the gene, and the significant diversity resulting from differences in gene copy number between individuals, coupled with structural variations such as deletions and insertions.

[0005] Patent Document 1 discloses a method for determining the KIR alleles present in an individual using a primer set specific to at least one exon of the KIR gene. Non-patent document 1 describes the successful implementation of KIR gene sequencing using the target capture method and next-generation sequencing (NGS), as well as high-precision KIR typing using specific statistical methods and AI technologies.

[0006] However, the conventional PCR method used in Patent Document 1 has fundamental drawbacks, such as requiring at least 60 ng of DNA for typing, limiting the number of samples that can be processed at once, and being unable to use fragmented DNA. Furthermore, the conventional PCR method has difficult-to-solve problems such as mis-base incorporation by DNA polymerase during PCR amplification, the production of chimeric molecules due to DNA elongation crossing over to other chromosomes (e.g., from maternal to paternal chromosomes) or other highly homologous genes, and the phenomenon of specific alleles not being amplified due to polymorphisms in genomic regions corresponding to PCR primers (allele drop). As a result, only 8 of the 16 KIR gene families could be analyzed. In addition, the method in Non-Patent Document 1 requires the use of a unique analysis algorithm and is not practical in terms of cost. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 5926183 [Non-patent literature]

[0008] [Non-Patent Document 1] Sakaue et al., Cell Genomics, Vol.2, 100101, March 9, 2022 [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention aims to overcome the technical challenges of conventional PCR-based typing methods and to provide a highly practical probe set and a typing method using the same that are suitable for efficiently and accurately typing multiple samples simultaneously using the sequence capture method. [Means for solving the problem]

[0010] To solve the aforementioned problems, the inventors conducted extensive research and, as a result, discovered that by using a probe set consisting of oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 1543, it is possible to simultaneously sequence the KIR gene in multiple samples, thus completing the present invention.

[0011] In other words, the present invention provides a KIR gene typing probe set consisting of oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 1543. Furthermore, the present invention provides a method for typing KIR genes using the probe set. [Effects of the Invention]

[0012] The probe set of the present invention has a base sequence designed relative to a reference sequence uniquely designed by the inventors, and is designed to comprehensively type the 16 genes included in the KIR region (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DP1, KIR3DS1, and KIR3DP1). Therefore, it becomes possible to simultaneously determine alleles of genes from multiple samples (determine allele names as named in the IMGT(registered trademark) / KIR database).

[0013] The sequencing capture method using the probe set of the present invention allows for the typing of even minute amounts of DNA, such as 1 ng. Furthermore, the typing method of the present invention enables the typing of fragmented DNA samples (e.g., cfDNA (cell-free DNA) or FFPE (formalin-fixed paraffin-embedded) samples), which was difficult with conventional methods using PCR. Therefore, the method of the present invention does not require high-quality DNA samples such as blood, and can be used to type from, for example, oral swab samples or swab-derived DNA, enabling non-invasive testing that does not burden the subject. Moreover, since the method of the present invention allows for the simultaneous processing of multiple samples, the cost per sample can be reduced (estimated to be about 1 / 6 compared to conventional methods using PCR), making it suitable for large-scale research and clinical applications. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows a schematic of a gene typing method using the sequence capture method according to the present invention. [Modes for carrying out the invention]

[0015] Human genome reference sequences are being updated sequentially, and "GRCh38 / hg38" is widely known as a relatively new reference sequence. Conventional probes were generally designed based on known reference sequences. The inventors have independently created a reference sequence that is not identical to any human genome sequence, but is similar to all human genome sequences, based on known reference sequences, and have designed 1543 novel probes that are effective in specifically capturing each gene in the KIR region using this unique reference sequence.

[0016] Each probe included in the probe set of the present invention is fundamentally based on having the base sequence shown in SEQ ID NOs: 1 to 1543, but may also have a base sequence in which one to several bases (or within 10%, preferably within 5%, more preferably within 3%, and even more preferably within 1%) of the bases constituting each probe are substituted, deleted, or inserted. However, this is limited to probes that exhibit the probe function intended in the present invention.

[0017] Furthermore, the present invention provides a method for typing KIR genes, including sequence capture using the above-described probe set. Figure 1 shows an overview of the typing method using the sequence capture method. Specifically, this method includes the following steps. (1) A process of obtaining DNA fragments by fragmenting the DNA contained in a sample obtained from a subject. (2) A step of mixing the DNA fragment obtained in step (1) with the probe set of the present invention and hybridizing it to obtain a DNA library. (3) A step of concentrating the DNA library obtained in step (2). (4) A step of desorbing the probe from the DNA of the concentrated DNA library and determining the sequence of the DNA library from which the probe has been desorbed using NGS.

[0018] (1) The step of fragmenting the DNA contained in the sample obtained from the subject is carried out using a conventional method in the art. For example, fragmentation of DNA using enzymatic cleavage is preferred. The ends of the fragmented DNA are repaired (smoothed) and adenylated as necessary. The amount of DNA contained in the sample may be 1 ng or more, preferably 5 ng or more, more preferably 10 ng or more. The upper limit of the DNA amount is not particularly limited, but can be, for example, less than 100 ng, less than 75 ng, less than 50 ng, less than 40 ng, less than 30 ng, or less than 15 ng, etc. When using a fragmented DNA sample such as cfDNA or FFPE as the sample, the step of further fragmenting the DNA may be omitted. As the sample, body fluids such as blood, or samples derived from non-invasively collected swabs, etc. can be used.

[0019] Next, an adapter is ligated, and different index sequences (up to 384 types) are added to each subject (specimen) by PCR to form a DNA library. At this time, it is preferable to measure the DNA fragment length and DNA concentration. The DNA libraries are mixed at an equimolar concentration to obtain a sample.

[0020] (2) Next, the DNA library obtained in (1) is mixed with the probe set of the present invention and hybridized. Before hybridization, it is necessary to add a label to each probe contained in the probe set that can bind to the label carried on a separation carrier such as beads in a subsequent enrichment step. As the substance for labeling the probe, biotin is preferably used. [[ID=ll]]

[0021] (3) Next, for example, the DNA library hybridized with the biotin-labeled probe is adsorbed onto streptavidin-immobilized beads (magnetic beads). The DNA fragments that did not hybridize with the probe are removed by washing, and the target DNA library adsorbed on the magnetic beads is concentrated.

[0022] (4) Finally, the concentrated DNA library and probe are separated using a standard method. The DNA library from which the probe has been removed is, if necessary, amplified by PCR using an adapter sequence and specific primers, and then the base sequences of up to 384 samples are determined using NGS. Subsequently, data analysis based on the base sequence information is performed.

[0023] The present invention will be explained below with specific examples. However, the following examples do not limit the scope of the present invention. Also, the number attached to "Sample" in the table below is a number that identifies the individual (subject or specimen) from whom the DNA sample was collected. "copy" represents the number of copy number variations detected. "X" means that the corresponding gene (allele) was not detected in the sample. [Examples]

[0024] Example 1 1543 probes (sequence numbers: 1 to 1543) of the present invention were synthesized. For 384 DNA samples containing 1 to 50 ng of DNA, the target DNA library was enriched using the sequence capture method with the obtained 1543 probes. After desorbing the probes, the DNA was sequenced using NGS.

[0025] The specific steps are as follows: A DNA library for sequencing the KIR gene was prepared using a commercially available kit. Specifically, the DNA was cut into fragments of approximately 300 bp using an enzyme, and then subjected to DNA end repair, adenine addition, and adapter ligation to form the DNA library. The adapters were adjusted to add 384 different index sequences, each unique to the sample, by PCR.

[0026] The ligated DNA libraries were analyzed for fragment length using a 4150 TapeStation system (Agilent), and DNA concentration was measured using an Invitorogen Qubit4 Fluorometer (Thermo Fisher Scientific). Based on the measurement data, 384 prepared DNA libraries were mixed at equimolar concentrations.

[0027] The probe of the present invention was labeled with biotin. Using reagents from xGen® Hybridization and Wash v2 Reagents (Integrated DNA Technologies), the probe was hybridized to the DNA library obtained above according to the protocol.

[0028] The biotin-avidin reaction was used to enrich the probe-bound DNA library. Following standard procedures, the probe was detached from the DNA library, and then PCR was performed using the adapter sequence and specific primers. The DNA libraries from the 384 obtained samples were sequenced using NextSeq2000 (Illumina) (using P1 reagent). The results are shown in Tables 1-48 below.

[0029] As shown in each table, the presence or absence of 16 types of KIR genes (alleles) in the DNA of each sample could be identified using the method described above. Furthermore, if the relevant gene was present, it was possible to type the first three digits of the seven digits following the asterisk (*) indicating the genotype, which identify alleles with amino acid substitutions, and the next two digits, which indicate base substitutions without amino acid mutations.

[0030] [Table 1]

[0031] [Table 2]

[0032] Table 3

[0033] Table 4

[0034] Table 5

[0035] Table 6

[0036] Table 7

[0037] Table 8

[0038] Table 9

[0039] Table 10

[0040] Table 11

[0041] Table 12

[0042] Table 13

[0043] Table 14

[0044] Table 15

[0045] Table 16

[0046] Table 17

[0047] Table 18

[0048] Table 19

[0049] Table 20

[0050] Table 21

[0051] Table 22

[0052] Table 23

[0053] Table 24

[0054] Table 25

[0055] Table 26

[0056] Table 27

[0057] Table 28

[0058] Table 29

[0059] Table 30

[0060] Table 31

[0061] Table 32

[0062] Table 33

[0063] Table 34

[0064] Table 35

[0065] Table 36

[0066] Table 37

[0067] Table 38

[0068] Table 39

[0069] Table 40

[0070] Table 41

[0071] Table 42

[0072] Table 43

[0073] [Table 44]

[0074] [Table 45]

[0075] [Table 46]

[0076] [Table 47]

[0077] [Table 48]

[0078] Example 2 (The present invention) Typing was performed using the capture method for the 24 DNA samples listed in Tables 49-53 below (TYGD001-TYGD024: containing 1-50 ng of DNA), in accordance with the method described in Example 1. Equipment used: NextSeq2000 (manufactured by Illumina)

[0079] Comparative Example 1 (Conventional Method) Typing using PCR (conventional method) was performed on 24 types of DNA samples (TYGD001 to TYGD024; each containing 60 ng of DNA) listed in Tables 49 to 53 below. Reagents used: NGSgo-Amp KIR (GenDX) Equipment used: NextSeq2000 (manufactured by Illumina) Methods and conditions: The procedure was carried out according to the manufacturer's protocol.

[0080] Tables 49-53 show the typing results for each sample DNA obtained using the conventional PCR method (Comparative Example 1) and the method of the present invention (Example 2). In the table, "TRUE" means that the typing results for Comparative Example 1 and Example 2 were the same. "Blank" indicates that typing of the corresponding gene was not possible.

[0081] As shown in Tables 49-53, the conventional method using PCR (Comparative Example 1) was unable to type the eight genes KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DP1, and KIR3DP1. On the other hand, for the remaining eight genes that could be typed using the conventional method (Comparative Example 1), the DNA typing results obtained using the conventional method (Comparative Example 1) and the method of the present invention (Example 2) were completely identical.

[0082] In other words, the method of the present invention makes it possible to type KIR genes, which could not be typed by conventional methods using PCR, and its accuracy was confirmed to be at least as high as that of conventional methods. In addition, the cost required for the method of the present invention is cheaper than that required for conventional methods, and the cost per sample in the method of the present invention was about 1 / 6 of that of conventional methods.

[0083] [Table 49]

[0084] [Table 50]

[0085] [Table 51]

[0086] [Table 52]

[0087] Table 53

Claims

1. A probe set for KIR gene typing, consisting of oligonucleotides, each having the nucleotide sequence shown in SEQ ID NOs: 1 to 1543.

2. The probe set according to claim 1, wherein biotin is added.

3. (1) A process of fragmenting DNA contained in a sample obtained from a subject to obtain DNA fragments, (2) A step of mixing the DNA fragment obtained in step (1) with the probe set described in claim 1 or 2 and hybridizing it to obtain a DNA library. (3) A step of concentrating the DNA library hybridized with the probe, (4) A step of removing the probe from the concentrated DNA library and determining the sequence of the obtained DNA library using NGS. A typing method for the KIR gene, including [specific gene].

4. The typing method according to claim 3, wherein the probe set is the probe set according to claim 2, and the concentration step is carried out by adding streptavidin-immobilized beads.

5. The typing method according to claim 3, further comprising the step of PCR amplification of the DNA fragment before sequencing the DNA fragment.

6. The typing method according to claim 4, further comprising the step of PCR amplification of a DNA fragment before sequencing of the DNA fragment.