Method and kit for genotyping of multi-system red blood cell (RBC) blood group based on next-generation sequencing (NGS)

Description

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims the benefit and priority of Chinese Patent Application No. 2024104330574, filed with the China National Intellectual Property Administration on Apr. 10, 2024, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.

REFERENCE TO SEQUENCE LISTING

A computer readable XML file entitled “Sequence Listing”, that was created on Dec. 26, 2024, with a file size of 364, 120 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure belongs to the technical field of blood group genotyping, and specifically relates to a method and a kit for genotyping of 25 coding genes in 17 red blood cell (RBC) blood group systems based on next-generation sequencing (NGS).

BACKGROUND

A human blood group system consists of red blood cell (RBC) surface antigens (proteins, glycoproteins, or glycolipids). The blood group system is inherited and controlled by a single gene or a gene cluster of two or three closely-linked homologous genes. As of July 2023, there are a total of 378 RBC blood group antigens recognized by the International Society of Blood Transfusion (ISBT), of which 360 belong to 45 blood group systems. Human RBCs selectively express antigens from different blood group systems, which constitute each person's unique blood group combination. In addition to ABO, there are 17 most clinically important RBC blood group systems in China, including H, RH (including RHD and RHCE), MNS, Kidd, Diego, Duffy, Lewis, Kell, P1Pk, GLOB, Lutheran, Yt, SC, DO, CO, LW, and JR.

To ensure the safety of clinical blood transfusion, a primary factor is that blood groups of the donor and the patient are consistent. There are two problems:

First, unconventional blood group systems cause adverse reactions to blood transfusions. Nowadays, ABO and RhD blood groups (conventional blood groups) are those that medical institutions conduct routine test and homotype transfusion; for blood group systems (non-conventional blood groups) other than the above two, routine test is omitted due to technical and cost limitations. Therefore, for patients with chronic blood transfusions, multiple transfusions of unconventional blood with an incompatible blood group may stimulate the immune system to produce blood group alloantibodies, triggering transfusion reactions that may even be life-threatening in severe cases. According to statistics from Serious Hazards of Transfusion (SHOT) in the UK, all hemolytic transfusion reactions in the past five years were caused by incompatible transfusions with unconventional blood group systems. A key to solving this problem lies in matching blood transfusions with unconventional blood group systems in patients with chronic blood transfusions.

Second, the supply of blood for rare blood groups should be guaranteed. A rare blood group does not refer to a specific blood group, but refers to a blood group whose frequency is less than one in a thousand among all people. For example, the above-mentioned 17 most clinically important blood group systems in China mainly include 31 key rare blood groups. Blood of rare blood groups is an extremely scarce resource. It is currently the only way to solve the problem of blood supply for clinical rare blood types by conducting large-scale screening among blood donors to find those with rare blood groups and freezing blood of the rare blood groups. However, the difficulty in screening rare blood groups is that there is no technology suitable for simultaneous detection of multiple blood group systems yet at home and abroad.

RBC blood group identification belongs to the field of in vitro diagnostics, and identification methods include serological test and genetic diagnosis. The serological test is the most traditional blood group identification method as well as a commonly used blood group identification method in clinical medical institutions. The RBC blood group is identified by specific reaction in vitro between serum (antibody reagent) containing known specific antibodies and the antigens on a RBC surface to be tested, showing the advantages of simple operation, rapidity, and easy interpretation. However, the serological test is limited by the availability of blood group antibody reagents, and most unconventional blood group systems do not have commercially available antibody reagents globally; a few commercially available reagents completely depend on imports, and are expensive and in short supply. This is a “bottleneck” faced by the current in vitro diagnostic industry of blood groups.

When the serological test was limited, RBC blood group genetic diagnosis based on PCR and sequencing came into being. There are 1,568 alleles listed in a blood group gene mutation database of the National Center for Biotechnology Information (NCBI). Blood type can be predicted by detecting the coding genes of blood group antigens and analyzing genetic polymorphism characteristics. The most significant advantage of genotyping to detect blood groups is to supplement and overcome the deficiencies of serology. For example, diseases, drugs, and treatments (especially recent blood transfusions) interfere with the serological test, making blood group results ambiguous; there are individuals with genetic mutations that result in reduced antigen expression or with unexpected antibodies. In addition, for rare blood groups for which serological test antibody reagents are not available, genotyping is the only way to predict the blood group.

The blood group genetic testing technologies currently widely used in clinical and blood collection and supply institutions in China are polymerase chain reaction-sequence specific primer (PCR-SSP) and first-generation sequencing (Sanger). A prerequisite for the PCR-SSP to identify blood group alleles is to clarify DNA sequences and single nucleotide polymorphisms (SNPs) in order to prepare corresponding primers or probes. Therefore, this technique is not suitable for detecting mutations of unknown sequences or blood group gene with high polymorphism. The Sanger is a gold standard for genetic testing, and can accurately obtain the nucleotide sequence of a target gene or a certain fragment and identify unknown mutations. However, this technique cannot directly distinguish between two haplotypes and may sometimes produce ambiguous typing results. Moreover, as low-to-medium-throughput genetic testing techniques, the PCR-SSP and Sanger are in a high cost and less efficient for screening rare blood groups. In this case, next-generation sequencing (NGS), also known as high-throughput sequencing, is more appropriate. The NGS determines a DNA sequence while synthesizing same by capturing newly synthesized end tags, and allows the detection of one single haplotype DNA sequence to avoid ambiguous typing results. In addition, the NGS has an extremely high detection throughput and can greatly increase the detection throughput, and is highly suitable for large-scale screening of rare blood groups.

SUMMARY

A purpose of the present disclosure is to provide a method and a kit for genotyping of 25 coding genes in 17 red blood cell (RBC) blood group systems based on next-generation sequencing (NGS). In the present disclosure, the method based on NGS and multiplex PCR effectively improves both accuracy and detection efficiency for the genotyping of RBC blood group, and can save the manpower in genotyping of the RBC blood group to a certain extent.

The present disclosure provides a primer set for simultaneously detecting genotyping of 17 RBC blood group systems, including primers with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 336.

Preferably, a first primer set includes the primers with the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 108;

• • a second primer set includes the primers with the nucleotide sequences shown in SEQ ID NO: 109 to SEQ ID NO: 222; and
  • a third primer set includes the primers with the nucleotide sequences shown in SEQ ID NO: 223 to SEQ ID NO: 336.

The present disclosure further provides a kit for simultaneously detecting genotyping of 17 RBC blood group systems, including the primer set and a PCR reaction solution.

Preferably, the first primer set, the second primer set, and the third primer set in the primer set are packaged independently.

The present disclosure further provides a method for simultaneously detecting genotyping of 17 RBC blood group systems, including the following steps: (1) subjecting three capture and amplification systems to first amplification separately to obtain three first amplification products, where the three capture and amplification systems are prepared by using a nucleic acid extracted from a sample as a template and a first primer set with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 108, a second primer set with nucleotide sequences shown in SEQ ID NO: 109 to SEQ ID NO: 222, and a third primer set with nucleotide sequences shown in SEQ ID NO: 223 to SEQ ID NO: 336, respectively;

(2) purifying the three first amplification products separately, and subjecting three resulting purified first amplification products to second amplification with a sequencing universal adapter carrying an index separately to obtain three second amplification products; and

(3) purifying and mixing the three second amplification products to allow sequencing to obtain the genotyping of the RBC blood group systems.

Preferably, the sample in step (1) is selected from the group consisting of a whole blood sample, a plasma sample, and a paraffin tissue sample.

Preferably, the three capture and amplification systems in step (1) each have a volume of 25 μL, and include 1 μL of a mixture of the first primer set or the second primer set or the third primer set, 3 μL of an enzyme mixture Pxp-1st-mix, 1 μL of the template, and nuclease-free water as a balance.

Preferably, a PCR reaction program of the first amplification in step (1) includes: initial denaturation at 98° C. for 20 min; 9 cycles of denaturation at 98° C. for 15 s, annealing at 62° C. for 20 min, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 5 min; and heat preservation at 10° C.

Preferably, a system of the second amplification in step (2) has a volume of 12.5 μL, and includes 6.5 μL of an enzyme mixture Pxp-2nd-mix, 4 μL for one of the three purified first amplification products, 1 μL of a universal adapter N5XX, and 1 μL of a universal adapter A7XX.

Preferably, a PCR reaction program of the second amplification in step (2) includes: initial denaturation at 98° C. for 2 min; 23 cycles of denaturation at 98° C. for 15 s, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 10 min; and heat preservation at 4° C.

Beneficial effects: the present disclosure provides a PCR primer set and a kit for genotyping and capturing 25 coding genes of 17 RBC blood group systems based on a MiSeq NGS platform of the Illumina platform. The primer and kit can simultaneously detect multiple samples and multiple genes, including hereditary mutations such as point mutations and short indels. The kit effectively improves detection efficiency and accuracy, while reducing costs, simplifying operating steps, and providing flexible packaging specifications.

In the present disclosure, a detection method for detecting 31 key rare blood groups of 17 RBC blood group systems is constructed based on the primer set or kit. High-throughput sequencing is conducted to simultaneously detect 17 RBC blood group system-related genes to improve detection efficiency and reduce costs. The high-throughput gene sequencing can break through the limitations of traditional detection technology and improve detection sensitivity. Meanwhile, variations including known mutation sites and unknown mutation sites can be detected to obtain comprehensive and accurate detection results. By sequencing 17 RBC blood group system-related genes and their mutation sites, specific gene sequence information can be obtained, thereby providing accurate and efficient detection for rare blood group detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow chart for RBC blood group identification using the primer set or kit of the present disclosure;

FIG. 2 shows an electrophoresis pattern of primer specificity verification in Example 1;

FIG. 3 shows an electrophoresis pattern of primer detection sensitivity verification in Example 2; and

FIG. 4 shows results of the repeatability verification of primer detection in Example 3.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure provides a primer set for simultaneously detecting genotyping of 17 RBC blood group systems, including primers with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 336.

In the present disclosure, the RBC blood group system and corresponding genetic information are shown in Table 1.

TABLE 1
Gene information of the 17 RBC blood group systems

								Number
		Blood			Number	Number	Number	of
		group			of	of	of	primer
No.	ISBT No.	system	Coding gene	Chromosome	sites	types	bases	pairs

1	ISBT 018	H	FUT1	chr19	75	57	58	8
			FUT2	chr19	31	37	32	6
			SLC35C1	chr11	6	4	3	3
2	ISBT 004	RH	RhD	chr1	249	387	231	12
			RhCE	chr1	130	183	136	10
3	ISBT 002	MNS	GYPA	chr4	45	44	123	3
			GYPB	chr4	24	15	14	3
4	ISBT 009	Kidd	SLC14A1(JK)	chr18	29	32	49	7
5	ISBT 010	Diego	SLC4A1(DI)	chr17	39	40	19	5
6	ISBT 008	Duffy	ACKR1(FY)	chr1	21	24	229	6
7	ISBT 007	Lewis	FUT3	chr19	42	60	38	6
			FUT6	chr19	37	20	21	5
			FUT7	chr9	2	2	1	1
8	ISBT 006	Kell	KEL	chr7	96	94	67	16
9	ISBT 003	P1Pk	A4GALT	chr22	39	41	198	7
10	ISBT 028	GLOB	B3GALNT1	chr3	14	14	15	6
11	ISBT 005	Lutheran	BCAM(LU)	chr19	50	42	332	11
			KLF1	chr19	58	56	148	9
			GATA1	chrX	1	1	1	1
12	ISBT 011	Yt	ACHE	chr7	4	4	4	2
13	ISBT 013	SC	ERMAP	chr1	7	6	8	3
14	ISBT 014	DO	ART4	chr12	19	14	22	7
15	ISBT 015	CO	AQP1	chr7	9	9	450	4
16	ISBT 016	LW	ICAM4	chr19	2	2	11	1
17	ISBT 032	JR	ABCG2	chr4	43	37	1946	26
Total					1072	1225	4156	168

The present disclosure is based on 1,072 blood group system typing sites of 25 genes shown in Table 1: H (ISBT 018), Rh (ISBT 004), MNS (ISBT 002), Kidd (JK, ISBT 009), Diego (DI, ISBT 010), Duffy (FY, ISBT 008), Lewis (ISBT 007), Kell (ISBT 006), P1Pk (ISBT 003), Globoside (GLOB, ISBT 028), Lutheran (LU, ISBT 005), YT (ISBT 011), Scianna (SC, ISBT 013), Dombrock (DO, ISBT 014), Colton (CO, ISBT 015), Landsteiner-Wiener (LW, ISBT 016), and Junior (JR, ISBT 032). The present disclosure develops a multiplex PCR-based RBC blood type NGS typing primer set, kit and method adapted to the Illumina sequencing platform. The primer set is designed based on the target region of the above gene. The target region preferably includes the full length of the coding region sequences of the 25 antigen-coding genes of the above-mentioned 17 blood group systems and the splicing flanking region within 100 bp to 150 bp. In the present disclosure, the 26 genes are preferably selected from the University of California Santa Cruz (UCSC) database; the typing sites have been disclosed in databases and literature, most of which are collected in the ISBT database, and a small number are derived from literature.

In the present disclosure, the specific primers in the primer set are completely complementary or partially complementary to the above-mentioned target region. Preferably, each pair of specific primers for the target region contains two parts, labeled as 3′-end Tag1 and 5′-end Tag2. A fragment sequence of the 3′-end Tag1 is completely complementary or partially complementary to the target region, and is preferably a nucleic acid molecule with a specific sequence, with an unlimited number of bases, preferably 20 to 40. The 5′-end Tag2 is used to combine with the universal primer during the amplification of the target region, thus completing the specific labeling of the amplification products of the target regions of different samples to be tested. The 5′-end Tag2 has preferably 3 to 20, more preferably 4 to 10 bases.

In the present disclosure, a total of 168 pairs of primers are designed based on the above genes and design principles. Each pair of primers contains mutation sites in the gene. The size of a target region amplified by each pair of primers is 150 bp to 300 bp, with wide coverage and multiple detection sites; the primers have a relatively uniform Tm value (57° C. to 63° C.); the primers show desirable specificity, stability, and uniformity during multiplex amplification, and have non-specific amplification. The primers preferably further include specific modifications, and the modified primers are not easily degraded by nucleases; specifically, the primer-specific modifications include one or more of thio modification, deoxyuracil modification, 5′-reverse dT modification, and 5′-amino modification. A thio-modified primer is a derivative in which the double-bonded oxygen atom on the oligonucleotide chain is replaced by a sulfur atom, and can resist the degradation of nuclease and enhance the stability of the primer. Each deoxythymine in the oligonucleotide sequence of a deoxyuracil-modified primer is replaced by deoxyuracil, and can increase the double-stranded dissolution temperature by 1.7° C., thereby increasing the stability of the double-stranded. The 5′ end of the oligonucleotide sequence in a 5′-reverse dT-modified primer contains a reverse dT, thereby forming a cross-link, which can inhibit the degradation of exonucleases during the extension of DNA polymerase. The 5′-amino modification includes C6 amino modification and C12 amino modification. The former can be used to connect some compounds that do not affect its function even near the oligonucleotide, while the latter is used to ligate the affinity purification group and some fluorescent marks.

In the present disclosure, according to the nature of each primer, such as the amplification interval, Tm value, primer sequence, and actual expansion during the later test, the primer set is divided into 3 pools, which are specifically shown in Table 2. The number marked after the sequence in Table 2 is the number of SEQ ID NO. For example, (1) indicates that the nucleotide sequence of a sequence is serialized as SEQ ID NO: 1. The primer set 1 includes: RB_018, RB_023, RB_026, RB_029, RB_032, RB_034, RB_037, RB_039, RB_063, RB_016, RB_053, RB_055, RB_057, RB_048, RB_051, RB_070, RB_064, RB_066, RB_068, RB_118, RB_123, RB_106, RB_108, RB_110, RB_013, RB_003, RB_093, RB_097, RB_099, RB_101, RB_104, RB_042, RB_126, RB_077, RB_079, RB_082, RB_085, RB_087, RB_089, RB_091, RB_125, RB_130, RB_134, RB_137, RB_144, RB_146, RB_149, RB_153, RB_155, RB_157, RB_160, RB_167, RB_138, RB_140, and their nucleotide sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 108 in sequence.

The primer set 2 includes: RB_020, RB_021, RB_022, RB_025, RB_027, RB_031, RB_035, RB_058, RB_060, RB_062, RB_015, RB_054, RB_046, RB_049, RB_052, RB_071, RB_072, RB_074, RB_065, RB_067, RB_069, RB_117, RB_120, RB_122, RB_105, RB_109, RB_112, RB_114, RB_010, RB_012, RB_002, RB_005, RB_007, RB_095, RB_098, RB_102, RB_041, RB_043, RB_045, RB_127, RB_081, RB_084, RB_088, RB_075, RB_131, RB_133, RB_136, RB_143, RB_147, RB_150, RB_152, RB_156, RB_158, RB_161, RB_163, RB_165, RB_141, and their nucleotide sequences are shown in SEQ ID NO: 109 to SEQ ID NO: 222 in sequence.

The primer set 3 includes: RB_019, RB_024, RB_028, RB_030, RB_033, RB_036, RB_038, RB_059, RB_061, RB_017, RB_056, RB_047, RB_050, RB_073, RB_116, RB_119, RB_121, RB_124, RB_107, RB_111, RB_113, RB_115, RB_009, RB_011, RB_014, RB_001, RB_004, RB_006, RB_008, RB_092, RB_094, RB_096, RB_100, RB_103, RB_040, RB_044, RB_076, RB_078, RB_080, RB_083, RB_086, RB_090, RB_128, RB_129, RB_132, RB_135, RB_142, RB_145, RB_148, RB_151, RB_154, RB_159, RB_162, RB_164, RB_166, RB_168, RB_139, and their nucleotide sequences are shown in SEQ ID NO: 223 to SEQ ID NO: 336 in sequence.

TABLE 2
Information of primer pairs

	Target gene
Primer ID	fragment	Sequence of forward primer	Sequence of reverse primer
RB_001	FUT1-1	AGTCTCCCTGGCTCTCAAGG (SEQ ID	CGATGGACAGGAGGCTACAC (SEQ ID
		NO: 273)	NO: 274)
RB_002	FUT1-2	CAGAGTCTGGCAGGGTGAAG (SEQ ID	TTTTCGTGGTCACCAGCAAC (SEQ ID
		NO: 169)	NO: 170)
RB_003	FUT1-3	GTGTAGCCTCCTGTCCATCG (SEQ ID	GTGGGGACTATCTGCAGGTT (SEQ ID
		NO: 51)	NO: 52)
RB_004	FUT1-4	TTGCTGGTGACCACGAAAAC (SEQ ID	CTTCCACCATCTCCGGGAAC (SEQ ID
		NO: 275)	NO: 276)
RB_005	FUT1-5	CACACTCTGCGCCTCTTCC (SEQ ID	TTTATCCTGCCTGCCATGCA (SEQ ID
		NO: 171)	NO: 172)
RB_006	FUT1-6	CTCAAGTCCGCGTACTCCTC (SEQ ID	CACCTGGACTGTCTACCCCA (SEQ ID
		NO: 277)	NO: 278)
RB_007	FUT1-7	CGTGGCATACTGTCCCATCT (SEQ ID	CTGGCCTTCCTGCTAGTCTG (SEQ ID
		NO: 173)	NO: 174)
RB_008	FUT1-8	CGGTCTGGACACAGGATCG (SEQ ID	CCTGGGACTAAGGAGTGCTG (SEQ ID
		NO: 279)	NO: 280)
RB_009	FUT2-1	GCCTCCATCTCCCAGCTAAC (SEQ ID	AGGCGGCCTATTGCATTGAT (SEQ ID
		NO: 267)	NO: 268)
RB_010	FUT2-2	CGATCAATGCAATAGGCCGC (SEQ ID	GGGATGTGGCGGTATTCCTC (SEQ ID
		NO: 165)	NO: 166)
RB_011	FUT2-3	CCTGGCAGAACTACCACCTG (SEQ ID	ATAGTCCCCTCGGCGAACAT (SEQ ID
		NO: 269)	NO: 270)
RB_012	FUT2-4	GAGGAGGCCCAGAAGTTCCT (SEQ ID	CAGCAAACACCACATCACCG (SEQ ID
		NO: 167)	NO: 168)
RB_013	FUT2-5	TCGTGGTCACCAGTAATGGC (SEQ ID	GTCGGGGAGGGTGTAATTGG (SEQ ID
		NO: 49)	NO: 50)
RB_014	FUT2-6	GCGGAGACACCATCTACCTG (SEQ ID	GAGGGAGGCAGAGAAGGAGA (SEQ ID
		NO: 271)	NO: 272)
RB_015	SLC35C1-1	AACCTCTGCCTCAAGTACGTC (SEQ ID	TGACCACTCTATCCCCCGTG (SEQ ID
		NO: 129)	NO: 130)
RB_016	SLC35C1-2	GAGGGCCGCAATACTCAGTC (SEQ ID	TTCGTGGTGTAGATGGCGTT (SEQ ID
		NO: 19)	NO: 20)
RB_017	SLC35C1-3	CATCGGCTACGTGACAGGAC (SEQ ID	TCTTCTCGCTGTCTTTGGGG (SEQ ID
		NO: 241)	NO: 242)
RB_018	RhD-1	GCCCTCAAGTAGGTGTTGGA (SEQ ID	CCCTGCTATTTGCTCCTGTGA (SEQ ID
		NO: 1)	NO: 2)
RB_019	RhD-2	AAATCTCGTCTGCTTCCCCC (SEQ ID	GATCCAGCCACCATCCCAAT (SEQ ID
		NO: 223)	NO: 224)
RB_020	RhD-3	AAGATCTGACCGTGATGGCG (SEQ ID	GAACCTGTCCTTTCGGGGTC (SEQ ID
		NO: 109)	NO: 110)
RB_021	RhD-4	TTCTCAGTCGTCCTGGCTCTC (SEQ ID	TTCAAAACCCTGGAAACCCCA (SEQ ID
		NO: 111)	NO: 112)
RB_022	RhD-5	GAGGATGCCGACACTCACTG (SEQ ID	TCAGCCCAAGTAGGAGACCA (SEQ ID
		NO: 113)	NO: 114)
RB_023	RhD-6	GACCTTTGGAGCAGGAGTGT (SEQ ID	GTCCTGTTAGACCCAAGTGCT (SEQ ID
		NO: 3)	NO: 4)
RB_024	RhD-7	CTCGAGGCTCAGACCTTTGG (SEQ ID	TTAGACCCAAGTGCTGCCCA (SEQ ID
		NO: 225)	NO: 226)
RB_025	RhD-8	GCTTCCTTTACCCACACGCT (SEQ ID	CCTTCAGCCAAAGCAGAGGA (SEQ ID
		NO: 115)	NO: 116)
RB_026	RhD-9	GTCTCACCTGCCAATCTGCT (SEQ ID	CCCAGCTAAGGACTCTGCAC (SEQ ID
		NO: 5)	NO: 6)
RB_027	RhD-10	CTGATGCCCAAGTGACCACC (SEQ ID	AGGAGATGGGGCACATAGAC (SEQ ID
		NO: 117)	NO: 118)
RB_028	RhD-11	TGAGATTAAAAATCCTGTGCTCCA (SEQ	GTTCCTCCTGCAATGCTCCT (SEQ ID
		ID NO: 227)	NO: 228)
RB_029	RhD-12	GGCTGTTTCAAGAGATCAAGCC (SEQ	TTCTCTGACTCCAGTGCCTG (SEQ ID
		ID NO: 7)	NO: 8)
RB_030	RhCE-13	TCTGTCTCTGACCTTGTTTCAT (SEQ ID	GGCTGTTTCAAGAGATCAAGCC (SEQ
		NO: 229)	ID NO: 230)
RB_031	RhCE-14	GAAAGGTGGCCTCACACTGA (SEQ ID	CCTGGCAATGGCACTACTGA (SEQ ID
		NO: 119)	NO: 120)
RB_032	RhCE-15	CCCAGCTAAGGACTCTGCAC (SEQ ID	GTCTCACCTGCCAATCTGCT (SEQ ID
		NO: 9)	NO: 10)
RB_033	RhCE-16	CTTCAGCCAAAGCAGAGAGC (SEQ ID	GCTGGTCACTTGCAGCAAGA (SEQ ID
		NO: 231)	NO: 232)
RB_034	RhCE-17	TTAGACCCAAGTGCTGCCCA (SEQ ID	CTCGAGGCTCAGACCTTTGG (SEQ ID
		NO: 11)	NO: 12)
RB_035	RhCE-18	CTCAGCCCAAGTATGAGACCA (SEQ ID	TTTCTCCAAGGACCATCAGGG (SEQ ID
		NO: 121)	NO: 122)
RB_036	RhCE-19	AATGGAGCTTTTGGCCCTTTTC (SEQ ID	TCAGTCATCCTGGCTCTCCTTC (SEQ ID
		NO: 233)	NO: 234)
RB_037	RhCE-20	GAACCTGTCCTTTCGGGGTC (SEQ ID	AAGATCTGACCGTGATGGCG (SEQ ID
		NO: 13)	NO: 14)
RB_038	RhCE-21	GATCCAGCCACCATCCCAAT (SEQ ID	AAATCTCGTCTGCTTCCCCC (SEQ ID
		NO: 235)	NO: 236)
RB_039	RhCE-22	CCTGCTATTTGCTCCTGTGA (SEQ ID	CTCAAGCCCTCAAGTAGGTGT (SEQ ID
		NO: 15)	NO: 16)
RB_040	GYPA-1	TGGTGTACAACATGATGGTTTGA (SEQ	TGTACAACAGGGTGAATGGAGT (SEQ
		ID NO: 291)	ID NO: 292)
RB_041	GYPA-2	CTCCTAGAGCTGTTCACACTGG (SEQ	AGGCAAGGTGATGTTATGCTGA (SEQ ID
		ID NO: 181)	NO: 182)
RB_042	GYPA-3	AGGCATTTGAAACAAGCAATGGA (SEQ	AAGGAAACCCGCAGAACAGT (SEQ ID
		ID NO: 63)	NO: 64)
RB_043	GYPA-4	GCAGTGACAGGTCCCCTAAA (SEQ ID	CTGCTCAGTCACCTCGTTCT (SEQ ID
		NO: 183)	NO: 184)
RB_044	GYPA-5	TGTCACGAAAGCTTGAGAACTG (SEQ	GCTCTATGTCCACGCAGTCA (SEQ ID
		ID NO: 293)	NO: 294)
RB_045	GYPA-6	GCAGTGACAGGTCCCCTAAA (SEQ ID	TTATGGTCCGCTCAGTCACC (SEQ ID
		NO: 185)	NO: 186)
RB_046	JK-1	TGGCTAATCTGGAGGGCTCT (SEQ ID	ACCTTTAAGCTGGTTGGCAAG (SEQ ID
		NO: 133)	NO: 134)
RB_047	JK-2	CTTCCAGACAAACCCGTGGT (SEQ ID	TGCCCACAGTAACTGGTCAG (SEQ ID
		NO: 245)	NO: 246)
RB_048	JK-3	GCAAGTGCAACCAAAGCTCA (SEQ ID	GGCATTTTGAAAACCAATTGTAACT
		NO: 27)	(SEQ ID NO: 28)
RB_049	JK-4	CAGCCTGCTTTGTCACATGC (SEQ ID	GCGAATGTGAGAAGCCAGTG (SEQ ID
		NO: 135)	NO: 136)
RB_050	JK-5	ACCAGTGGGAGTTGGTCAGA (SEQ ID	CCCATTGGTCCCTAGGAAGC (SEQ ID
		NO: 247)	NO: 248)
RB_051	JK-6	CCTGAGTTCTGACCCCTCCT (SEQ ID	CAGAACCCTGGCCCTGATTT (SEQ ID
		NO: 29)	NO: 30)
RB_052	JK-7	GTAATCAGGGCACTGTGCATTC (SEQ	TGGACTTCAGGAGCATTTCCC (SEQ ID
		ID NO: 137)	NO: 138)
RB_053	DI-1	GCCACTCACACACTGAAGCTC (SEQ ID	TGGGGTGTGATAGGCACTGAC (SEQ ID
		NO: 21)	NO: 22)
RB_054	DI-2	ATGAAGACCAGCAGAGCAGG (SEQ ID	AGTTCCCAAGTGCCTCCAAC (SEQ ID
		NO: 131)	NO: 132)
RB_055	DI-3	GCTGTTCTTGAACTTGCGCA (SEQ ID	GGTATTTTCCAGCCCAAGCC (SEQ ID
		NO: 23)	NO: 24)
RB_056	DI-4	CAACACCACCAGCAGGATGA (SEQ ID	CCCTGCTGGTGTTTGAGGAA (SEQ ID
		NO: 243)	NO: 244)
RB_057	DI-5	TGCACTGCAGTGGAGATCAG (SEQ ID	ACTCTTGCCTCTGACCCTCT (SEQ ID
		NO: 25)	NO: 26)
RB_058	FY-1	CCATCCTGGTCTCTTGGTGC (SEQ ID	AGGCCATCAGAGTTACACCG (SEQ ID
		NO: 123)	NO: 124)
RB_059	FY-2	ATGGCCTCCTCTGGGTATGT (SEQ ID	AGCATGAAGAGGACAGTGCT (SEQ ID
		NO: 237)	NO: 238)
RB_060	FY-3	GCACTGCCCTTCTTCATCCT (SEQ ID	GCTGAGCCATACCAGACACA (SEQ ID
		NO: 125)	NO: 126)
RB_061	FY-4	TCTTCCGCTGGCAGCTCT (SEQ ID NO:	CCACTCCCCAAATTCCCACA (SEQ ID
		239	NO: 240)
RB_062	FY-5	CACTGTAGCCTGTCTTGCCA (SEQ ID	AAAATTGCCAGGGCTTCTGC (SEQ ID
		NO: 127)	NO: 128)
RB_063	FY-6	TTGTCAACATGTCTGGCCCA (SEQ ID	AAGAAACCACCCGCTTCACA (SEQ ID
		NO: 17)	NO: 18)
RB_064	FUT3-1	GAGAGAGGGTTGGCCACAAA (SEQ ID	AGGAGCTGGACAAGGACCA (SEQ ID
		NO: 33)	NO: 34)
RB_065	FUT3-2	TGGTCCTTGTCCAGCTCCT (SEQ ID NO:	CCAAGGGGACCATGATGGAG (SEQ ID
		145)	NO: 146)
RB_066	FUT3-3	ACAGCGTCTCCATCATGGTC (SEQ ID	CAGCGACTCCGACATCTTCA (SEQ ID
		NO: 35)	NO: 36)
RB_067	FUT3-4	CTGAGTCCGGCTTCCAGTTG (SEQ ID	CCAACCCTAAGTCACGCCTC (SEQ ID
		NO: 147)	NO: 148)
RB_068	FUT3-5	GCGTGACTTAGGGTTGGACA (SEQ ID	TTCTCCTACCTGCGTGTGTC (SEQ ID
		NO: 37)	NO: 38)
RB_069	FUT3-6	TGGAAAGGCCATGTCCGTAG (SEQ ID	ACTCTGACCCATGGATCCCC (SEQ ID
		NO: 149)	NO: 150)
RB_070	FUT6-1	GCGTGTCTGGTACCTGGATT (SEQ ID	CCCAGCAGAAGCAACTACGA (SEQ ID
		NO: 31)	NO: 32)
RB_071	FUT6-2	TCGTAGTTGCTTCTGCTGGG (SEQ ID	GGAACCATGATGGAGACGCT (SEQ ID
		NO: 139)	NO: 140)
RB_072	FUT6-3	GTCTTGGCCGAGAGGTTGAG (SEQ ID	TCATGTACAACCCCAGTGCC (SEQ ID
		NO: 141)	NO: 142)
RB_073	FUT6-4	TTGGGGACTCCATGCTGAAC (SEQ ID	ACAAACCCATAGCTCTGCCC (SEQ ID
		NO: 249)	NO: 250)
RB_074	FUT6-5	ATGGGTTTGTTAAAAGGCCACG (SEQ	TCTCCCCACTTCCCAGATACT (SEQ ID
		ID NO: 143)	NO: 144)
RB_075	FUT7-7	GTGGGTGTGGCTAGGAGACT (SEQ ID	TCACCATCCTTGTCTGGCAC (SEQ ID
		NO: 195)	NO: 196)
RB_076	KEL-1	ACAGCGGAAATACCTGGCAA (SEQ ID	CACTTGATCCCCTGGTTCCC (SEQ ID
		NO: 295)	NO: 296)
RB_077	KEL-2	GGGACTTCCATGAGCTTCAGT (SEQ ID	GGGTTTTGGGTACTGTGTGGA (SEQ ID
		NO: 67)	NO: 68)
RB_078	KEL-3	CCAACGTCTGCAGCATTCTCT (SEQ ID	ATGCTCCTGGGAGCTGATTC (SEQ ID
		NO: 297)	NO: 298)
RB_079	KEL-4	CTCCCTTGTGGTCTTCCCTT (SEQ ID	CTCCCTTGTGGTCTTCCCTT (SEQ ID
		NO: 69)	NO: 70)
RB_080	KEL-5	ATCATAACACCTGTCGGCCC (SEQ ID	GGGAGGGACTGTGTAGGTCT (SEQ ID
		NO: 299)	NO: 300)
RB_081	KEL-6	CATACCTGTGTTGGGGGTGA (SEQ ID	TGGTCCCATTGGTGTTTGTCA (SEQ ID
		NO: 189)	NO: 190)
RB_082	KEL-7	GGAGGGTCAGAGAAGTGACGA (SEQ	GCCACTGGGCTGTATACTCA (SEQ ID
		ID NO: 71)	NO: 72)
RB_083	KEL-8	ATCCCCATCTCGCTTGTTCC (SEQ ID	AGCCCTTTTCCAAGGGTCAG (SEQ ID
		NO: 301)	NO: 302)
RB_084	KEL-9	TGCCCAATTCCCCAATCACA (SEQ ID	TCCTGAACAGGAGCCACTCA (SEQ ID
		NO: 191)	NO: 192)
RB_085	KEL-10	AATACACCCGCTCCTCTCCT (SEQ ID	GGAGCTGCCTTCACGAGTAT (SEQ ID
		NO: 73)	NO: 74)
RB_086	KEL-11	GCGGCGAACCTCTGCTTTAG (SEQ ID	ATCCCCTCCCCCAGTTAGC (SEQ ID NO:
		NO: 303)	304)
RB_087	KEL-12	GAGAGGAAGATCCCCATGCC (SEQ ID	CCTTCCTCCAGATCTTTCGGG (SEQ ID
		NO: 75)	NO: 76)
RB_088	KEL-13	GTCTCCTCCCAGCAAGGTTC (SEQ ID	CCAGTCTCTCTTGTGCCCAG (SEQ ID
		NO: 193)	NO: 194)
RB_089	KEL-14	GTCCAACTGTGTCTTCGCCA (SEQ ID	AGAGCCGATCCAGACAATGG (SEQ ID
		NO: 77)	NO: 78)
RB_090	KEL-15	AGCATCTTCCACCCTGCTTT (SEQ ID	AGCCCTTGTCTTTTTGCCTCT (SEQ ID
		NO: 305)	NO: 306)
RB_091	KEL-16	GGCTGACTAGGTTAGGGGGT (SEQ ID	TCACCTCTTGGTTCCTCCCA (SEQ ID
		NO: 79)	NO: 80)
RB_092	A4GALT-1	GGGCCCCTCACAAGTACATT (SEQ ID	CTGAGGCCTTCTACCCCATC (SEQ ID
		NO: 281)	NO: 282)
RB_093	A4GALT-2	ATGGGGTAGAAGGCCTCAGG (SEQ ID	TCTCAAGAACCTGCGGAACC (SEQ ID
		NO: 53)	NO: 54)
RB_094	A4GALT-3	CCGTTGTAGTGGTCCACGAA (SEQ ID	CTGGGAGCCCTACCTGCT (SEQ ID NO:
		NO: 283)	284)
RB_095	A4GALT-4	CATGAGTGCGATCCTGGAGG (SEQ ID	TTCATGTGCTCGGTGGAGTC (SEQ ID
		NO: 175)	NO: 176)
RB_096	A4GALT-5	CGGAAGCCCTTTCATCAGGA (SEQ ID	ATCTACTGGCACGTTGTGGG (SEQ ID
		NO: 285)	NO: 286)
RB_097	A4GALT-6	CCCACAACGTGCCAGTAGAT (SEQ ID	TTGGCTCTGGCTGATGTTCA (SEQ ID
		NO: 55)	NO: 56)
RB_098	A4GALT-7	CCTCCTCCCCACTGCGAG (SEQ ID NO:	CGCAAGGGCTCTGGGGAC (SEQ ID NO:
		177)	178)
RB_099	B3GALNT1-1	GTAAGCCAGCACACTGACCT (SEQ ID	GCAGCCCATGGCTTTTCTTC (SEQ ID
		NO: 57)	NO: 58)
RB_100	B3GALNT1-2	GGGCTGCAATCACACGTCTC (SEQ ID	TCCTTTCAAGGTGTTCCCTCC (SEQ ID
		NO: 287)	NO: 288)
RB_101	B3GALNT1-3	TCCTTGGCACCAAATCTCTGG (SEQ ID	GCCCCAATGCCAAGTACGTA (SEQ ID
		NO: 59)	NO: 60)
RB_102	B3GALNT1-4	TCAGTGTCTGTCTTCATTACGT (SEQ ID	CCAGGCAGGCCATTAGAGTT (SEQ ID
		NO: 179)	NO: 180)
RB_103	B3GALNT1-5	GGCATTGGGGCAAAACTCAG (SEQ ID	TGACCTCCCACCCTTCAGAT (SEQ ID
		NO: 289)	NO: 290)
RB_104	B3GALNT1-6	ATCTGAAGGGTGGGAGGTCA (SEQ ID	TCTCTGGACTGTCCTTCCGA (SEQ ID
		NO: 61)	NO: 62)
RB_105	LU-1	AGCTCAGTTGCTCTCTTGCA (SEQ ID	AACAAGGAGTGTGGCTTGGT (SEQ ID
		NO: 157)	NO: 158)
RB_106	LU-2	AGCTGCAGAGAGAAAGGACC (SEQ ID	CACACGTAGTCTCGCTCGTC (SEQ ID
		NO: 43)	NO: 44)
RB_107	LU-3	CTGAGATGCAGGGCTCTGAG (SEQ ID	GGATGCCCGAGGACACTTAC (SEQ ID
		NO: 259)	NO: 260)
RB_108	LU-4	CGTGTTTGGTAAGTGTCCTCG (SEQ ID	CTGTCCCCTCCTCCTCCAG (SEQ ID NO:
		NO: 45)	46)
RB_109	LU-5	CGACTTCAGAGTCCCAGCTC (SEQ ID	GCTGCTCACCTGGGTTCAT (SEQ ID NO:
		NO: 159)	160)
RB_110	LU-6	CGATCTCTCCCAGAGGGCTA (SEQ ID	CTCATGAGGTGTGGAGCCTG (SEQ ID
		NO: 47)	NO: 48)
RB_111	LU-7	CCTTAGATCCCACGGAGCAC (SEQ ID	CAGATCAGGTGGCTGCCTAA (SEQ ID
		NO: 261)	NO: 262)
RB_112	LU-8	CCATGCTGTCGCTCAGTTCT (SEQ ID	CGAGCAGAAGATGGAGTCCC (SEQ ID
		NO: 161)	NO: 162)
RB_113	LU-9	CTGGACAAACAGGACGAGTTC (SEQ ID	CTCCAGCTGAGTTTGGGGTC (SEQ ID
		NO: 263)	NO: 264)
RB_114	LU-10	CTGATCGGAGCCTCCATAGC (SEQ ID	CTCACTGCAGGGACAGGATG (SEQ ID
		NO: 163)	NO: 164)
RB_115	LU-11	GCACCGGTGAGTGACTGAG (SEQ ID	TCATTGCAGATAGCAGGCCAC (SEQ ID
		NO: 265)	NO: 266)
RB_116	KLF1-1	CCCATCCCCAGTCACTAGGA (SEQ ID	GGACATGACTGGGCAGACAG (SEQ ID
		NO: 251)	NO: 252)
RB_117	KLF1-2	CCTCTGCAACCCTTCTTCCC (SEQ ID	GACTGCAGAGGATCCAGGTG (SEQ ID
		NO: 151)	NO: 152)
RB_118	KLF1-3	TCTCGGCTATCACACCTGGA (SEQ ID	GCTCCTCGGGTGGCTACTTC (SEQ ID
		NO: 39)	NO: 40)
RB_119	KLF1-4	TACCCGGACAGTAGCCCGTA (SEQ ID	GGCTTTTGGGTTCGGAGGAT (SEQ ID
		NO: 253)	NO: 254)
RB_120	KLF1-5	GGAAGTAGCCACCCGAGGAG (SEQ ID	CGAGACTCTGGGCGCATATG (SEQ ID
		NO: 153)	NO: 154)
RB_121	KLF1-6	ATCCTCCGAACCCAAAAGCC (SEQ ID	CCCTCCACGTGAAGTCTGAG (SEQ ID
		NO: 255)	NO: 256)
RB_122	KLF1-7	GAGGAGATCCAGGTCCCAGG (SEQ ID	GACAGGCAAACAAGACCCCT (SEQ ID
		NO: 155)	NO: 156)
RB_123	KLF1-8	CCCCAAGATCTGTGACTGTGG (SEQ ID	CCCTCGAAGGGGCTATCACA (SEQ ID
		NO: 41)	NO: 42)
RB_124	KLF1-9	GGTCCAGGTGCTGGGTAAAA (SEQ ID	TGATAGCAGCCTCCAACGTC (SEQ ID
		NO: 257)	NO: 258)
RB_125	GATA1-1	CCTTTCCCTGGACCCCTACT (SEQ ID	ACACACCCACAATTTCAGGACT (SEQ
		NO: 81)	ID NO: 82)
RB_126	YT-1	TAGACCCATGGTGGCTTTCC (SEQ ID	CCTTCGTGCCTGTGGTAGAT (SEQ ID
		NO: 65)	NO: 66)
RB_127	YT-2	ACACTCTGGAAGGTTGTAGCG (SEQ ID	TCCTTCTCCTCCTCCTCTGG (SEQ ID
		NO: 187)	NO: 188)
RB_128	ERMAP-1	TGCTTGGCAGGCAGTATCTT (SEQ ID	CCCTGACAGCCTTTTCCAGT (SEQ ID
		NO: 307)	NO: 308)
RB_129	ERMAP-2	CTCCCAGTTGGCCTTGTCTC (SEQ ID	TCTCTCACTAGCACCGTCCT (SEQ ID
		NO: 309)	NO: 310)
RB_130	ERMAP-3	GGATGGGAAGGACCAGGATG (SEQ ID	TTGCCACAAATGACCCTGGG (SEQ ID
		NO: 83)	NO: 84)
RB_131	ART4-1	TGCTCAGGTTCCCAGTTGAC (SEQ ID	CTACACAGGGGCCACCATTC (SEQ ID
		NO: 197)	NO: 198)
RB_132	ART4-2	GAATGGTGGCCCCTGTGTAG (SEQ ID	TAGAGCCATGGCCTCTGTTG (SEQ ID
		NO: 311)	NO: 312)
RB_133	ART4-3	AGCTGGATTGCTGAGGTGAG (SEQ ID	AAAAGCCCACTTAGCCTGGC (SEQ ID
		NO: 199)	NO: 200)
RB_134	ART4-4	GGCCATGGCTCTAGTAAAGTCA (SEQ	TCGACTTCGACTTCGCACC (SEQ ID NO:
		ID NO: 85)	86)
RB_135	ART4-5	TTAAGCCAGGCTAAGTGGGC (SEQ ID	AAATCTGCAACCACATTCACCA (SEQ ID
		NO: 313)	NO: 314)
RB_136	ART4-6	CGGGTGAATGCTCTGTTGGA (SEQ ID	TCAGGATGAAGCTGCAAGGG (SEQ ID
		NO: 201)	NO: 202)
RB_137	ART4-7	TCAGTCTCATCCGTAACCGT (SEQ ID	GACTTCGGCATTCCCCTGAA (SEQ ID
		NO: 87)	NO: 88)
RB_138	AQP1-1	CCGGCCCTATAAATAGGCCC (SEQ ID	CGACACCTTCACGTTGTCCT (SEQ ID
		NO: 105)	NO: 106)
RB_139	AQP1-2	GGGCTTCAAATACCCGGTGG (SEQ ID	GGTGATGCCTGAGAGGATGG (SEQ ID
		NO: 335)	NO: 336)
RB_140	AQP1-3	CCGTGCCCTCATGTACATCA (SEQ ID	TCTCTACGTGACCTCCAGCA (SEQ ID
		NO: 107)	NO: 108)
RB_141	AQP1-4	TTCTCCCTCCAACCTCTCCC (SEQ ID	GGTCAGGCTTACCATGGGAC (SEQ ID
		NO: 221)	NO: 222)
RB_142	ICAM4-1	GCTCAATTGCAGCAACAGCT (SEQ ID	GCCCCTGTCCCTCACTGTA (SEQ ID NO:
		NO: 315)	316)
RB_143	ABCG2-1	CACCTGCATTCCTTGGCTCT (SEQ ID	CTGACCTCGTAATCCACCCG (SEQ ID
		NO: 203)	NO: 204)
RB_144	ABCG2-2	CGGCCTCCCAAAGTACTAGG (SEQ ID	CGAGCAAAAGGGGAAAAGCC (SEQ ID
		NO: 89)	NO: 90)
RB_145	ABCG2-3	GCGGGTTCAAGCGATTCTAC (SEQ ID	CCTAGTACTTTGGGAGGCCG (SEQ ID
		NO: 317)	NO: 318)
RB_146	ABCG2-4	GCTCAGGATCTCAGGATGCG (SEQ ID	GTGCTGTGCCCACTCAAAAG (SEQ ID
		NO: 91)	NO: 92)
RB_147	ABCG2-5	GTTTGCACCAGGGCATCATT (SEQ ID	GCCTTTGGTTAAGACCGAGC (SEQ ID
		NO: 205)	NO: 206)
RB_148	ABCG2-6	TCTCTAGGACTGAGAAGGGAGT (SEQ	AACCCACTGAGCACAGAAGT (SEQ ID
		ID NO: 319)	NO: 320)
RB_149	ABCG2-7	TGGCAGTGCAGGTTTCTCTC (SEQ ID	GTTTCATGTTGGCCAGGCTG (SEQ ID
		NO: 93)	NO: 94)
RB_150	ABCG2-8	ATCTTTTGGCTTCCCTGGGC (SEQ ID	CTTGTCCAACGTGCATGTGG (SEQ ID
		NO: 207)	NO: 208)
RB_151	ABCG2-9	TGGAGTTGCACTCTACCTCA (SEQ ID	TGAAAGCCCATGGATCAACCT (SEQ ID
		NO: 321)	NO: 322)
RB_152	ABCG2-10	CGGGGAAGCCATTGGTGTTT (SEQ ID	AGTTGTGCCTGTCTTCCCAT (SEQ ID
		NO: 209)	NO: 210)
RB_153	ABCG2-11	AGGCCACGTGATTCTTCCAC (SEQ ID	AGGCTCCTTTAAGGAACAGTGG (SEQ
		NO: 95)	ID NO: 96)
RB_154	ABCG2-12	CATTCGCGCACAACTCACTT (SEQ ID	TGTTTACCTTGCCCTGCTCC (SEQ ID
		NO: 323)	NO: 324)
RB_155	ABCG2-13	AGCTTGGGAATGCAGTCACA (SEQ ID	TGGTAGGGACTTGAAGAGGGT (SEQ ID
		NO: 97)	NO: 98)
RB_156	ABCG2-14	GCAAACACAGTTCAGACTCACC (SEQ	TCCCAAACATACGGTGACCT (SEQ ID
		ID NO: 211)	NO: 212)
RB_157	ABCG2-15	CTCTGACCTGCTGCTATGGC (SEQ ID	ACAACATTGGAGACCGAGGG (SEQ ID
		NO: 99)	NO: 100)
RB_158	ABCG2-16	GGTGCAACTGACTTCACCCA (SEQ ID	AACAGACAAGTCTAGCCTGCC (SEQ ID
		NO: 213)	NO: 214)
RB_159	ABCG2-17	GAGCTATAGAGGCCTGGGGA (SEQ ID	ATCCACTGATTGCAAAGCCAC (SEQ ID
		NO: 325)	NO: 326)
RB_160	ABCG2-18	AGCCACCACATTGCTAAACT (SEQ ID	TAGCCTTACCTCCCTCACCC (SEQ ID
		NO: 101)	NO: 102)
RB_161	ABCG2-19	AGTATCCCAAGGCCTCCTGA (SEQ ID	CAAAGTCAGGCTGAACTAGAGC (SEQ
		NO: 215)	ID NO: 216)
RB_162	ABCG2-20	TCAGGAGCAAAAGGACAGCA (SEQ ID	TCTTACAGGACTGGCACACG (SEQ ID
		NO: 327)	NO: 328)
RB_163	ABCG2-21	ACCTTGGAGTCTGCCACTTT (SEQ ID	AAGGATGATGTTGTGATGGGCA (SEQ ID
		NO: 217)	NO: 218)
RB_164	ABCG2-22	GTTGTTGCAAGCCGAAGAGC (SEQ ID	CAGGCTTTGCAGACATCTATGG (SEQ ID
		NO: 329)	NO: 330)
RB_165	ABCG2-23	TCAGCCAAAGCACTTACCCA (SEQ ID	TATAGCATGTGTTGGAGGGAAA (SEQ ID
		NO: 219)	NO: 220)
RB_166	ABCG2-24	AGAACCAGACCTGACATGCG (SEQ ID	CATGAAACCTGGTCTCAACGC (SEQ ID
		NO: 331)	NO: 332)
RB_167	ABCG2-25	GCCAGTTTCTTGGAAATAGCCA (SEQ	GGAAACACCAATGGCTTCCC (SEQ ID
		ID NO: 103)	NO: 104)
RB_168	ABCG2-26	CGGGGAAGCCATTGGTGTTT (SEQ ID	AGTTGTGCCTGTCTTCCCAT (SEQ ID
		NO: 333)	NO: 334)

The present disclosure further provides a kit for simultaneously detecting genotyping of 17 RBC blood group systems, including the primer set, and a PCR reaction solution and a quality control.

In the present disclosure, the first primer set, the second primer set, and the third primer set in the primer set are packaged independently. The PCR reaction solution is preferably PCR Buffer, high-fidelity DNA polymerase, and nuclease-free water. In the examples, enzyme mixtures Pxp-1st-mix and Pxp-2nd-mix that mix the PCR Buffer with the high-fidelity DNA polymerase are selected, and the enzyme mixtures Pxp-1st-mix and Pxp-2nd-mix each preferably include commercial ingredients, such as main components in the enzyme mixture: high-fidelity enzyme and 5×HF PCR Buffer: Thermo Fisher scientific's “Phusion Hot Start II DNA Polymerase (2 U/μL)”, F565L; dNTP (dATP, dTTP, dCTP, dGTP): Sangon (BBI) A620046, A670046, A640046, 660046.

In the present disclosure, the quality control is preferably a human normal genomic DNA.

The present disclosure further provides a method for detecting genotyping of 17 RBC blood group systems, including the following steps as shown in FIG. 1: (1) subjecting three capture and amplification systems to first amplification separately to obtain three first amplification products, where the three capture and amplification systems are prepared by using a nucleic acid extracted from a sample as a template and a first primer set, a second primer set, and a third primer set, respectively;

(2) purifying the three first amplification products separately, and subjecting three resulting purified first amplification products to second amplification with a sequencing universal adapter carrying an index separately to obtain three second amplification products; and

(3) purifying and mixing the three second amplification products to allow sequencing to obtain the genotyping of the RBC blood group systems.

In the present disclosure, three capture and amplification systems are subjected to first amplification separately to obtain three first amplification products, where the three capture and amplification systems are prepared by using a nucleic acid extracted from a sample as a template and a first primer set, a second primer set, and a third primer set, respectively. Preferably, the sample is extracted using magnetic beads, the concentration and purity are measured, and the nucleic acid is quantitatively diluted. Since nano-scale superparamagnetic carboxyl magnetic beads can reversibly adsorb nucleic acid molecules under different salt concentrations and hydrophobic environments, the nucleic acid molecules can be specifically adsorbed based on this. High-purity nucleic acid samples can be obtained through subsequent washing and elution. Concentration and purity are measured using Qubit or NanoDrop. According to a measured nucleic acid sample concentration, the nucleic acid sample is diluted to 50-250 ng/μL with 10 mM Tris-HCl, preferably 100-200 ng/μL serves as a starting concentration for amplification and library construction.

In the examples of the present disclosure, a quality of the diluted nucleic acid is preferably further evaluated by 1.5% agarose gel electrophoresis at 150 V for 30 min. The quality of the extracted nucleic acid is determined based on the uniformity and integrity of the agarose gel electrophoresis results. High integrity or slight degradation of the nucleic acid does not affect the library construction. The extracted nucleic acids are grouped and labeled separately after passing the concentration measurement, purity measurement, and agarose gel electrophoresis test. The sample is selected from the group consisting of a whole blood sample, a plasma sample, and a paraffin tissue sample. Nucleic acids are extracted from the above samples and diluted to the starting concentration for amplification and library construction. 3 tubes of capture and amplification system are constructed according to the grouping of the pools, where a primer pair of each pool is regarded as 1 tube. The three capture and amplification systems each have a volume of 25 L, and include preferably 1 μL of a mixture of the first primer set or the second primer set or the third primer set, 3 μL of an enzyme mixture Pxp-1st-mix, 1 μL of the template, and nuclease-free water as a balance. In the mixture of each pool primer set, 26 μL of Tris-HCl and 124 μL of primer set are preferably used to form a pool 1 primer with a total volume of 150 μL and a final concentration of 200 nM; except that the primers GATA1_001, KLF1_004, KLF1_008, and LU_004 are all 2 μL, and the primer Rh_020 is 5 μL, the other primers are all 1 μL. 32 μL of Tris-HCl and 168 μL of primer set are used to form a pool 2 primer with a total volume of 200 μL and a final concentration of 150 nM; except that the primer A4GALT_007 is 6 μL, the primers ABCG2_019, ABCG2_023, FUT_007, KLF1_005, KLF1_007, LU_001, LU_005, LU_008, MNS_002, and Rh_018 are 2 μL, the primer YT_002 is 3 μL, and the primer Rh_010 is 11 μL, the other primers are all 1 μL. 8 μL of Tris-HCl and 142 μL of primer set are used to form a pool 3 primer with a total volume of 150 μL and a final concentration of 200 nM; except that the primers A4GALT_005, ABCG2_006, ABCG2_024, KLF1_003, KLF1_006, LU_007, MNS_001, Rh_011, Rh_013, and SLC35C1_003 are 2 μL, the primer Rh_021 is 5 μL, the other primers are all 1 μL. When constructing each pool primer solution, each primer in each pool primer set has a concentration of 30 μM.

In the present disclosure, the three capture and amplification systems are preferably subjected to first amplification to obtain three first amplification products (pre-library products). Preferably, a PCR reaction program of the first amplification includes: initial denaturation at 98° C. for 20 min; 9 cycles of denaturation at 98° C. for 15 s, annealing at 62° C. for 20 min, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 5 min; and heat preservation at 10° C.

In the present disclosure, the three first amplification products are purified separately, and three resulting purified pre-library products are subjected to second amplification with a sequencing universal adapter carrying an index separately to obtain three second amplification products (library construction products). The purification preferably includes purifying the PCR amplification products (pre-library products) of each sample using purification magnetic beads, removing the remaining primers, gDNA, and dNTPs in the PCR products and recovering the amplification products.

In the present disclosure, after the purification is completed, sequencing universal adapters (N5XX and A7XX) carrying an index are used to further PCR-amplify the purified products. A reaction system of the PCR amplification is calculated as 12.5 μL, and preferably includes: enzyme mixture Pxp-2nd-mix 6.5 μL; pre-library product 4 μL; universal adapter N5XX 1 μL, universal adapter A7XX 1 μL, with a total volume of 12.5 μL. The same sample includes 3 pre-library products, which are added to 3 tubes of PCR master mix containing the same universal adapter. The PCR reaction program is preferably: initial denaturation at 98° C. for 2 min; 23 cycles of denaturation at 98° C. for 15 s, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 10 min; and heat preservation at 4° C.

In the present disclosure, after the PCR products are obtained, the library construction products of each sample are preferably separately purified using purification magnetic beads. The remaining primers and dNTPs are removed from the PCR product and the amplification products are recovered; the concentration and purity are measured, and the sample quality is tested by 1.5% agarose gel electrophoresis. After passing the test, the sample is diluted and mixed at equal concentrations, and then sequenced uniformly. The RBC blood group is determined based on sequencing results (Table 3). The mixed sample refers to mixing the products of the same sample amplified and purified in the first step using three sets of primers, and the purified products amplified in the second step using adapters carrying the same index. The sample mixing requirements: different samples are amplified in the second step using adapters carrying different indexes; 3 pools of the same sample are amplified in the second step using adapters with the same index.

TABLE 3
Polymorphisms of each gene

	Nucleotide	Initiation	Termination		Nucleotide	Initiation	Termination
Gene	change	site	site	Gene	change	site	site

RHD	c.1G	25272548	25272548	GYPB	270 + 5T	143997545	143997545
RHD	c.3A	25272550	25272550	GYPB	270 + 5A	143997545	143997545
RHD	c.8G	25272555	25272555	GYPB	c.251G	143997559	143997559
RHD	c.17T	25272564	25272564	GYPB	c.236G	143997574	143997574
RHD	c.19T	25272566	25272566	GYPB	c.230T	143997580	143997580
RHD	c.26A	25272573	25272573	GYPB	c.208T	143997602	143997602
RHD	c.28T	25272575	25272575	GYPB	c.173C	143999413	143999413
RHD	c.29A	25272576	25272576	GYPB	c.173G	143999413	143999413
RHD	c.29C	25272576	25272576	GYPB	c.161G	143999425	143999425
RHD	c.41G	25272588	25272588	GYPB	c.161A	143999425	143999425
RHD	c.41T	25272588	25272588	GYPB	c.143T	143999443	143999443
RHD	c.48C	25272595	25272595	GYPB	c.143C	143999443	143999443
RHD	c.52G	25272599	25272599	GYPB	c.72G	144001249	144001249
RHD	c.53C	25272600	25272600	GYPB	c.72T	144001249	144001249
RHD	c.62C	25272609	25272609	GYPB	c.71A	144001250	144001250
RHD	c.65A	25272612	25272612	GYPB	c.71G	144001250	144001250
RHD	c.67C	25272614	25272614	GYPB	c.67A	144001254	144001254
RHD	c.67T	25272614	25272614	GYPB	c.67T	144001254	144001254
RHD	c.68A	25272615	25272615	GYPB	c.65C	144001256	144001256
RHD	c.73T	25272620	25272620	GYPB	c.65G	144001256	144001256
RHD	c.91A	25272638	25272638	GYPB	c.60A	144001261	144001261
RHD	c.92C	25272639	25272639	GYPB	c.60G	144001261	144001261
RHD	c.93_94insT	25272640	25272641	GYPB	c.59T	144001262	144001262
RHD	c.120A	25272667	25272667	GYPB	c.59G	144001262	144001262
RHD	c.130_	25272677	25272679	FUT3	c.1067A	5843773	5843773
	132delCTC			FUT3	c.1060G	5843780	5843780
RHD	c.147del	25272694	25272694	FUT3	c.1029G	5843811	5843811
RHD	148 + 1a	25272696	25272696	FUT3	c.980A	5843860	5843860
RHD	148 + 5c	25272700	25272700	FUT3	c.975A	5843865	5843865
RHD	c.150C	25284574	25284574	FUT3	c.974T	5843866	5843866
RHD	c.157T	25284581	25284581	FUT3	c.968C	5843872	5843872
RHD	c.161C	25284585	25284585	FUT3	c.962A	5843878	5843878
RHD	c.163C	25284587	25284587	FUT3	c.882T	5843958	5843958
RHD	c.173T	25284597	25284597	FUT3	c.858G	5843982	5843982
RHD	c.176A	25284600	25284600	FUT3	c.808G	5844032	5844032
RHD	c.178C	25284602	25284602	FUT3	c.808A	5844032	5844032
RHD	c.182C	25284606	25284606	FUT3	c.760A	5844080	5844080
RHD	c.182T	25284606	25284606	FUT3	c.735C	5844105	5844105
RHD	c.186G	25284610	25284610	FUT3	c.732T	5844108	5844108
RHD	c.186T	25284610	25284610	FUT3	c.667A	5844173	5844173
RHD	c.200G	25284624	25284624	FUT3	c.655A	5844185	5844185
RHD	c.201A	25284625	25284625	FUT3	c.612G	5844228	5844228
RHD	c.203A	25284627	25284627	FUT3	c.571A	5844269	5844269
RHD	c.203C	25284627	25284627	FUT3	c.560T	5844280	5844280
RHD	c.208T	25284632	25284632	FUT3	c.548T	5844292	5844292
RHD	c.209A	25284633	25284633	FUT3	c.522A	5844318	5844318
RHD	c.220G	25284644	25284644	FUT3	c.508A	5844332	5844332
RHD	c.223T	25284647	25284647	FUT3	c.484G	5844356	5844356
RHD	c.227A	25284651	25284651	FUT3	c.484A	5844356	5844356
RHD	c.242C	25284666	25284666	FUT3	c.478T	5844362	5844362
RHD	c.251C	25284675	25284675	FUT3	c.451G	5844389	5844389
RHD	c.254G	25284678	25284678	FUT3	c.370G	5844470	5844470
RHD	c.254T	25284678	25284678	FUT3	c.314T	5844526	5844526
RHD	c.260A	25284684	25284684	FUT3	c.304A	5844536	5844536
RHD	c.287A	25284711	25284711	FUT3	c.258T	5844582	5844582
RHD	c.301A	25284725	25284725	FUT3	c.202C	5844638	5844638
RHD	c.307C	25284731	25284731	FUT3	c.179G	5844661	5844661
RHD	c.329C	25284753	25284753	FUT3	c.179A	5844661	5844661
RHD	c.329T	25284753	25284753	FUT3	c.104A	5844736	5844736
RHD	336 − 2del	25290639	25290639	FUT3	c.61T	5844779	5844779
RHD	c.340G	25290645	25290645	FUT3	c.59G	5844781	5844781
RHD	c.340T	25290645	25290645	FUT3	c.55A	5844785	5844785
RHD	c.341A	25290646	25290646	FUT3	c.47C	5844793	5844793
RHD	c.346A	25290651	25290651	FUT3	c.41A	5844799	5844799
RHD	c.346C	25290651	25290651	FUT3	c.13G	5844827	5844827
RHD	c.347T	25290652	25290652	FUT3	c.13A	5844827	5844827
RHD	c.359A	25290664	25290664	FUT6	c.1002C	5831566	5831566
RHD	c.365T	25290670	25290670	FUT6	c.1002G	5831566	5831566
RHD	c.374A	25290679	25290679	FUT6	c.977G	5831591	5831591
RHD	c.376C	25290681	25290681	FUT6	c.977A	5831591	5831591
RHD	c.379T	25290684	25290684	FUT6	c.971C	5831597	5831597
RHD	c.394A	25290699	25290699	FUT6	c.971T	5831597	5831597
RHD	c.395A	25290700	25290700	FUT6	c.945C	5831623	5831623
RHD	c.399T	25290704	25290704	FUT6	c.945A	5831623	5831623
RHD	c.410A	25290715	25290715	FUT6	c.907C	5831661	5831661
RHD	c.410C	25290715	25290715	FUT6	c.907G	5831661	5831661
RHD	c.410T	25290715	25290715	FUT6	c.879C	5831689	5831689
RHD	c.413G	25290718	25290718	FUT6	c.879T	5831689	5831689
RHD	c.446A	25290751	25290751	FUT6	c.855G	5831713	5831713
RHD	c.455A	25290760	25290760	FUT6	c.855A	5831713	5831713
RHD	c.455C	25290760	25290760	FUT6	c.739G	5831829	5831829
RHD	c.458C	25290763	25290763	FUT6	c.739A	5831829	5831829
RHD	c.485G	25290790	25290790	FUT6	c.738C	5831830	5831830
RHD	486 + 1a	25290792	25290792	FUT6	c.738T	5831830	5831830
RHD	486 + 2a	25290793	25290793	FUT6	c.730C	5831838	5831838
RHD	c.490A	25300949	25300949	FUT6	c.730G	5831838	5831838
RHD	c.492A	25300951	25300951	FUT6	c.729T	5831839	5831839
RHD	c.494G	25300953	25300953	FUT6	c.729C	5831839	5831839
RHD	c.497C	25300956	25300956	FUT6	c.527G	5832041	5832041
RHD	c.505C	25300964	25300964	FUT6	c.527T	5832041	5832041
RHD	c.509C	25300968	25300968	FUT6	c.499_501insC	5832067	5832069
RHD	c.509G	25300968	25300968	FUT6	c.376C	5832192	5832192
RHD	c.510A	25300969	25300969	FUT6	c.376T	5832192	5832192
RHD	c.510T	25300969	25300969	FUT6	c.370C	5832198	5832198
RHD	c.513A	25300972	25300972	FUT6	c.370T	5832198	5832198
RHD	c.514T	25300973	25300973	FUT6	c.336C	5832232	5832232
RHD	c.520A	25300979	25300979	FUT6	c.336A	5832232	5832232
RHD	c.525A	25300984	25300984	FUT6	c.172G	5832396	5832396
RHD	c.535C	25300994	25300994	FUT6	c.172A	5832396	5832396
RHD	c.539A	25300998	25300998	FUT6	c.63G	5832505	5832505
RHD	c.539C	25300998	25300998	FUT6	c.63A	5832505	5832505
RHD	c.542C	25301001	25301001	FUT6	c.18G	5832550	5832550
RHD	c.544A	25301003	25301003	FUT6	c.18A	5832550	5832550
RHD	c.560C	25301019	25301019	FUT7	c.329G	137031410	137031410
RHD	c.579C	25301038	25301038	FUT7	c.329A	137031410	137031410
RHD	c.594T	25301053	25301053	KEL	c.2107A	142941344	142941344
RHD	c.602G	25301061	25301061	KEL	c.2030G	142942441	142942441
RHD	c.605T	25301064	2530106	KEL	c.2027A	142942444	142942444
RHD	c.607G	25301066	25301066	KEL	c.2024G	142942447	142942447
RHD	c.611C	25301070	25301070	KEL	c.2024A	142942447	142942447
RHD	c.621C	25301080	25301080	KEL	c.2023T	142942448	142942448
RHD	c.634A	25301093	25301093	KEL	c.1975del	142942496	142942496
RHD	c.634C	25301093	25301093	KEL	c.1947G	142942524	142942524
RHD	c.634T	25301093	25301093	KEL	c.1868G	142942948	142942948
RHD	634 + 5t	25301098	25301098	KEL	c.1868A	142942948	142942948
RHD	635 − 2c	25301518	25301518	KEL	c.1790C	142943026	142943026
RHD	635 − 2g	25301518	25301518	KEL	c.1790T	142943026	142943026
RHD	c.635A	25301520	25301520	KEL	c.1763G	142943284	142943284
RHD	c.640T	25301525	25301525	KEL	c.1757G	142943290	142943290
RHD	c.658C	25301543	25301543	KEL	c.1719T	142943328	142943328
RHD	c.661A	25301546	25301546	KEL	c.1678G	142943511	142943511
RHD	c.661T	25301546	25301546	KEL	c.1664A	142943525	142943525
RHD	c.667G	25301552	25301552	KEL	c.1643A	142943546	142943546
RHD	c.668C	25301553	25301553	KEL	c.1643G	142943546	142943546
RHD	c.671G	25301556	25301556	KEL	c.1596A	142943593	142943593
RHD	c.674T	25301559	25301559	KEL	c.1546T	142943829	142943829
RHD	c.676C	25301561	25301561	KEL	c.1490T	142944324	142944324
RHD	c.677A	25301562	25301562	KEL	c.1481T	142944333	142944333
RHD	c.680C	25301565	25301565	KEL	c.1481A	142944333	142944333
RHD	c.684_686del	25301569	25301571	KEL	c.1477T	142944337	142944337
RHD	c.686A	25301571	25301571	KEL	c.1475G	142944339	142944339
RHD	c.689T	25301574	25301574	KEL	c.1475A	142944339	142944339
RHD	c.697A	25301582	25301582	KEL	c.1420T	142944394	142944394
RHD	c.697C	25301582	25301582	KEL	c.1391C	142944665	142944665
RHD	c.700T	25301585	25301585	KEL	c.1391T	142944665	142944665
RHD	c.704C	25301589	25301589	KEL	c.1377A	142944679	142944679
RHD	c.705_707del	25301590	25301592	KEL	c.1298T	142946223	142946223
RHD	c.712A	25301597	25301597	KEL	c.1283G	142946238	142946238
RHD	c.722T	25301607	25301607	KEL	c.1283T	142946238	142946238
RHD	c.727A	25301612	25301612	KEL	c.1271C	142946250	142946250
RHD	c.728G	25301613	25301613	KEL	c.1271T	142946250	142946250
RHD	c.730C	25301615	25301615	KEL	c.1268T	142946253	142946253
RHD	c.731T	25301616	25301616	KEL	c.1217G	142946304	142946304
RHD	c.733C	25301618	25301618	KEL	c.1217A	142946304	142946304
RHD	c.739C	25301624	25301624	KEL	c.1216T	142946305	142946305
RHD	c.740G	25301625	25301625	KEL	c.1145G	142952567	142952567
RHD	c.744T	25301629	25301629	KEL	c.1145A	142952567	142952567
RHD	c.751C	25301636	25301636	KEL	c.1088A	142952624	142952624
RHD	c.758A	25301643	25301643	KEL	c.1042T	142953839	142953839
RHD	c.766C	25301651	25301651	KEL	c.986T	142953895	142953895
RHD	c.770T	25301655	25301655	KEL	c.986C	142953895	142953895
RHD	c.780A	25301665	25301665	KEL	c.965C	142953916	142953916
RHD	c.785del	25301670	25301670	KEL	c.965T	142953916	142953916
RHD	c.787A	25301672	25301672	KEL	c.948A	142953933	142953933
RHD	c.809A	25303329	25303329	KEL	924 + 1a	142954184	142954184
RHD	c.809G	25303329	25303329	KEL	924 + 1t	142954184	142954184
RHD	c.818A	25303338	25303338	KEL	c.913G	142954195	142954195
RHD	c.818T	25303338	25303338	KEL	c.913A	142954195	142954195
RHD	c.819A	25303339	25303339	KEL	c.905T	142954203	142954203
RHD	c.826C	25303346	25303346	KEL	c.905C	142954203	142954203
RHD	c.830A	25303350	25303350	KEL	c.903del	142954205	142954205
RHD	c.833A	25303353	25303353	KEL	c.877C	142954231	142954231
RHD	c.835A	25303355	25303355	KEL	c.877T	142954231	142954231
RHD	c.838A	25303358	25303358	KEL	c.875A	142954233	142954233
RHD	c.842G	25303362	25303362	KEL	c.875G	142954233	142954233
RHD	c.845A	25303365	25303365	KEL	c.842A	142954266	142954266
RHD	c.848T	25303368	25303368	KEL	c.841T	142954267	142954267
RHD	c.851T	25303371	25303371	KEL	c.841C	142954267	142954267
RHD	c.854A	25303374	25303374	KEL	c.787A	142954321	142954321
RHD	c.871T	25303391	25303391	KEL	c.780G	142954328	142954328
RHD	c.872G	25303392	25303392	KEL	c.780T	142954328	142954328
RHD	c.874C	25303394	25303394	KEL	c.758A	142954350	142954350
RHD	c.880C	25303400	25303400	KEL	c.758G	142954350	142954350
RHD	c.881T	25303401	25303401	KEL	c.745G	142954363	142954363
RHD	c.884A	25303404	25303404	KEL	c.745A	142954363	142954363
RHD	c.884C	25303404	25303404	KEL	c.743A	142954365	142954365
RHD	c.885T	25303405	25303405	KEL	c.743G	142954365	142954365
RHD	c.895G	25303415	25303415	KEL	c.742C	142954366	142954366
RHD	c.916A	25303436	25303436	KEL	c.742T	142954366	142954366
RHD	c.919A	25303439	25303439	KEL	736 − 1c	142954371	142954371
RHD	c.922T	25303442	25303442	KEL	c.730del	142954470	142954470
RHD	c.932G	25303452	25303452	KEL	c.715T	142954485	142954485
RHD	c.938T	25303458	25303458	KEL	c.578T	142957921	142957921
RHD	c.953A	25306609	25306609	KEL	c.578C	142957921	142957921
RHD	c.957A	25306613	25306613	KEL	c.578G	142957921	142957921
RHD	c.968T	25306624	25306624	KEL	c.574T	142957925	142957925
RHD	c.983A	25306639	25306639	KEL	c.539G	142957960	142957960
RHD	c.993delC	25306649	25306649	KEL	c.539A	142957960	142957960
RHD	c.993G	25306649	25306649	KEL	c.539C	142957960	142957960
RHD	c.998A	25306654	25306654	KEL	c.538T	142957961	142957961
RHD	c.1006C	25306662	25306662	KEL	526 − 2g	142957975	142957975
RHD	c.1012G	25306668	25306668	KEL	c.389G	142960939	142960939
RHD	c.1013C	25306669	25306669	KEL	c.389A	142960939	142960939
RHD	c.1015A	25306671	25306671	KEL	c.388C	142960940	142960940
RHD	c.1016A	25306672	25306672	KEL	c.388T	142960940	142960940
RHD	c.1018A	25306674	25306674	KEL	c.382T	142960946	142960946
RHD	c.1019T	25306675	25306675	KEL	c.306A	142961022	142961022
RHD	c.1025C	25306681	25306681	KEL	c.246A	142961082	142961082
RHD	c.1034A	25306690	25306690	KEL	c.230T	142961098	142961098
RHD	c.1048C	25306704	25306704	KEL	223 + 1a	142961359	142961359
RHD	c.1048G	25306704	25306704	KEL	c.184_185insT	142961398	142961399
RHD	c.1057A	25306713	25306713	A4GALT	c.631G	42693321	42693321
RHD	c.1059G	25306715	25306715	A4GALT	c.631C	42693321	42693321
RHD	c.1060A	25306716	25306716	A4GALT	c.241_243del	42693709	42693711
RHD	c.1061A	25306717	25306717	A4GALT	c.903G	42693049	42693049
RHD	c.1063A	25306719	25306719	A4GALT	c.903C	42693049	42693049
RHD	c.1070A	25306726	25306726	A4GALT	c.287A	42693665	42693665
RHD	c.1073C	25306729	25306729	A4GALT	c.299T	42693653	42693653
RHD	c.1107C	25317033	25317033	A4GALT	c.301del	42693651	42693651
RHD	c.1121A	25317047	25317047	A4GALT	c.418_428delins	42693524	42693534
RHD	c.1132G	25317058	25317058	A4GALT	c.470_496delins	42693456	42693482
RHD	c.1136T	25317062	25317062	A4GALT	c.473A	42693479	42693479
RHD	c.1145C	25317071	25317071	A4GALT	c.504dupC	42693447	42693449
RHD	c.1148C	25317074	25317074	A4GALT	c.914T	42693038	42693038
RHD	c.1152C	25317078	25317078	A4GALT	c.548A	42693404	42693404
RHD	1154 − 8a	25321881	25321881	A4GALT	c.987G	42692965	42692965
RHD	c.1154C	25321889	25321889	A4GALT	c.987A	42692965	42692965
RHD	c.1168G	25321903	25321903	A4GALT	c.559C	42693393	42693393
RHD	c.1169C	25321904	25321904	A4GALT	c.560A	42693392	42693392
RHD	c.1170C	25321905	25321905	A4GALT	c.656T	42693296	42693296
RHD	c.1177C	25321912	25321912	A4GALT	c.657del	42693295	42693295
RHD	c.1184T	25321919	25321919	A4GALT	c.732dupG	42693219	42693221
RHD	c.1187G	25321922	25321922	A4GALT	c.751T	42693201	42693201
RHD	c.1193T	25321928	25321928	A4GALT	c.752T	42693200	42693200
RHD	c.1194C	25321929	25321929	A4GALT	c.796del	42693156	42693156
RHD	c.1195A	25321930	25321930	A4GALT	c.783A	42693169	42693169
RHD	c.1199C	25321934	25321934	A4GALT	c.972_997del	42692955	42692980
RHD	c.1199T	25321934	25321934	A4GALT	c.1029dupC	42692922	42692924
RHD	c.1200T	25321935	25321935	A4GALT	c.201dupC	42693750	42693752
RHD	c.1203A	25321938	25321938	A4GALT	c.418T	42693534	42693534
RHD	c.1207T	25321942	25321942	A4GALT	c.498A	42693454	42693454
RHD	c.1208T	25321943	25321943	A4GALT	c.68dupT	42693883	42693885
RHD	c.1210C	25321945	25321945	A4GALT	c.290T	42693662	42693662
RHD	c.1212A	25321947	25321947	A4GALT	c.902del	42693050	42693050
RHD	c.1213G	25321948	25321948	A4GALT	c.388dupA	42693563	42693565
RHD	c.1215C	25321950	25321950	A4GALT	c.367C	42693585	42693585
RHD	c.1219_1224del	25321954	25321959	A4GALT	c.547_548del	42693404	42693405
RHD	c.1221A	25321956	25321956	A4GALT	c.480_	42693457	42693472
RHD	c.1222C	25321957	25321957		495dupGGCCGTGCAGGGGCGC
RHD	c.1224C	25321959	25321959	A4GALT	Exon 1 Deletion	42720797	42720870
RHD	c.1226T	25321961	25321961	B3GALNT1	c.376G	161086379	161086379
RHD	c.1227A	25321962	25321962	B3GALNT1	c.376A	161086379	161086379
RHD	c.1228G	25328898	25328898	B3GALNT1	c.202T	161086553	161086553
RHD	c.1229C	25328899	25328899	B3GALNT1	c.292_293insA	161086462	161086463
RHD	c.1238G	25328908	25328908	B3GALNT1	c.433T	161086322	161086322
RHD	c.1241T	25328911	25328911	B3GALNT1	c.537_538insA	161086217	161086218
RHD	c.1248_	25328918	25328919	B3GALNT1	c.648C	161086107	161086107
	1249insG			B3GALNT1	c.797C	161085958	161085958
RHD	c.1250C	25328920	25328920	B3GALNT1	c.811A	161085944	161085944
RHD	c.1252_	25328922	25328923	B3GALNT1	c.959A	161085796	161085796
	1253insT			B3GALNT1	c.203del	161086552	161086552
RHD	c.1252A	25328922	25328922	B3GALNT1	c.456G	161086299	161086299
RHCE	c.1254C	25362427	25362427	B3GALNT1	c.449G	161086306	161086306
RHCE	1228 − 2g	25362455	25362455	B3GALNT1	c.598del	161086157	161086157
RHCE	c.1154C	25362527	25362527	LU	c.99_104del	44811241	44811246
RHCE	c.1132C	25375370	25375370	LU	c.123insGG	44811265	44811267
RHCE	c.1132G	25375370	25375370	LU	Exons 3	44812163	44812391
RHCE	c.1130T	25375372	25375372		Deletion
RHCE	c.1118T	25375384	25375384	LU	c.212G	44812170	44812170
RHCE	1074 − 2g	25375428	25375428	LU	c.212A	44812170	44812170
RHCE	c.1044_	25385740	25385747	LU	c.223C	44812181	44812181
	1050dupGCTTCAT			LU	c.223T	44812181	44812181
RHCE	c.1025T	25385759	25385759	LU	c.230A	44812188	44812188
RHCE	c.1007A	25385777	25385777	LU	c.230G	44812188	44812188
RHCE	c.1007T	25385777	25385777	LU	c.282C	44812240	44812240
RHCE	c.1006G	25385778	25385778	LU	c.282G	44812240	44812240
RHCE	c.1006T	25385778	25385778	LU	c.326G	44812284	44812284
RHCE	c.966_	25385816	25385818	LU	c.326A	44812284	44812284
	968delinsC			LU	c.340G	44812298	44812298
RHCE	c.963del	25385821	25385821	LU	c.340A	44812298	44812298
RHCE	c.941C	25385843	25385843	LU	c.361T	44812319	44812319
RHCE	c.938delC	25388977	25388977	LU	c.419A	44812377	44812377
RHCE	c.919A	25388996	25388996	LU	Exons 4	44812478	44812548
RHCE	c.916G	25388999	25388999		Deletion
RHCE	c.908A	25389007	25389007	LU	c.469G	44812513	44812513
RHCE	c.907del	25389008	25389008	LU	c.469A	44812513	44812513
RHCE	c.890C	25389025	25389025	LU	c.524G	44813269	44813269
RHCE	c.872T	25389043	25389043	LU	c.524A	44813269	44813269
RHCE	c.827A	25389088	25389088	LU	c.524T	44813269	44813269
RHCE	c.818C	25389097	25389097	LU	c.611T	44813544	44813544
RHCE	c.818T	25389097	25389097	LU	c.611A	44813544	44813544
RHCE	c.807A	25389108	25389108	LU	c.662C	44813595	44813595
RHCE	801 + 1a	25390748	25390748	LU	c.662T	44813595	44813595
RHCE	c.800A	25390750	25390750	LU	c.679C	44813612	44813612
RHCE	c.787G	25390763	25390763	LU	c.679T	44813612	44813612
RHCE	c.774A	25390776	25390776	LU	c.691T	44813624	44813624
RHCE	c.748A	25390802	25390802	LU	c.711C	44813644	44813644
RHCE	c.744C	25390806	25390806	LU	c.711T	44813644	44813644
RHCE	c.744T	25390806	25390806	LU	c.711A	44813644	44813644
RHCE	c.734C	25390816	25390816	LU	c.714C	44813647	44813647
RHCE	c.733C	25390817	25390817	LU	c.714T	44813647	44813647
RHCE	c.733G	25390817	25390817	LU	c.824C	44814191	44814191
RHCE	c.730A	25390820	25390820	LU	c.824T	44814191	44814191
RHCE	c.728G	25390822	25390822	LU	c.905C	44814272	44814272
RHCE	c.722T	25390828	25390828	LU	c.905T	44814272	44814272
RHCE	c.712A	25390838	25390838	LU	c.1274A	44818820	44818820
RHCE	c.712G	25390838	25390838	LU	c.1274C	44818820	44818820
RHCE	c.697C	25390853	25390853	LU	c.1289C	44818835	44818835
RHCE	c.697G	25390853	25390853	LU	c.1289T	44818835	44818835
RHCE	c.695C	25390855	25390855	LU	c.1340C	44819059	44819059
RHCE	c.689C	25390861	25390861	LU	c.1340T	44819059	44819059
RHCE	c.685_687del	25390863	25390865	LU	c.1495C	44819367	44819367
RHCE	c.679_683del	25390867	25390871	LU	c.1495T	44819367	44819367
RHCE	c.676C	25390874	25390874	LU	c.1615A	44819487	44819487
RHCE	c.676del	25390874	25390874	LU	c.1615G	44819487	44819487
RHCE	c.676G	25390874	25390874	LU	c.1742A	44819705	44819705
RHCE	c.674G	25390876	25390876	LU	c.1742T	44819705	44819705
RHCE	c.667T	25390883	25390883	KLF1	c.1071A	12884903	12884903
RHCE	c.662C	25390888	25390888	KLF1	c.1048T	12884926	12884926
RHCE	c.662G	25390888	25390888	KLF1	c.1045del	12884929	12884929
RHCE	c.659A	25390891	25390891	KLF1	c.1040A	12884934	12884934
RHCE	c.649C	25390901	25390901	KLF1	c.1022A	12884952	12884952
RHCE	634 + 1t	25391993	25391993	KLF1	c.1004C	12884970	12884970
RHCE	c.602C	25392026	25392026	KLF1	c.1002del	12884972	12884972
RHCE	c.554A	25392074	25392074	KLF1	c.1001T	12884973	12884973
RHCE	c.538C	25392090	25392090	KLF1	c.994G	12884980	12884980
RHCE	c.527T	25392101	25392101	KLF1	c.991G	12884983	12884983
RHCE	c.526A	25392102	25392102	KLF1	c.991T	12884983	12884983
RHCE	c.520A	25392108	25392108	KLF1	c.983T	12884991	12884991
RHCE	c.512G	25392116	25392116	KLF1	c.983A	12884991	12884991
RHCE	c.506A	25392122	25392122	KLF1	c.977G	12884997	12884997
RHCE	c.506C	25392122	25392122	KLF1	c.973A	12885001	12885001
RHCE	c.501A	25392127	25392127	KLF1	c.968G	12885006	12885006
RHCE	c.500A	25392128	25392128	KLF1	c.954dupG	12885019	12885021
RHCE	c.500T	25392128	25392128	KLF1	c.948del	12885026	12885026
RHCE	c.497T	25392131	25392131	KLF1	c.947A	12885027	12885027
RHCE	c.494C	25392134	25392134	KLF1	c.946A	12885028	12885028
RHCE	c.491G	25392137	25392137	KLF1	c.939A	12885035	12885035
RHCE	487 − 5g	25392146	25392146	KLF1	914 − 1c	12885061	12885061
RHCE	486 + 5a	25402591	25402591	KLF1	c.902ins T	12885327	12885329
RHCE	486 + 1a	25402595	25402595	KLF1	c.899C	12885331	12885331
RHCE	c.473A	25402609	25402609	KLF1	c.895T	12885335	12885335
RHCE	c.464G	25402618	25402618	KLF1	c.887C	12885343	12885343
RHCE	c.461C	25402621	25402621	KLF1	c.874T	12885356	12885356
RHCE	460G	25402622	25402622	KLF1	c.868C	12885362	12885362
RHCE	c.455A	25402627	25402627	KLF1	c.826G	12885404	12885404
RHCE	c.380T	25402702	25402702	KLF1	c.826T	12885404	12885404
RHCE	c.377G	25402705	25402705	KLF1	c.809A	12885421	12885421
RHCE	c.375G	25402707	25402707	KLF1	c.802T	12885428	12885428
RHCE	c.374A	25402708	25402708	KLF1	c.796T	12885434	12885434
RHCE	c.365C	25402717	25402717	KLF1	c.663del	12885567	12885567
RHCE	c.365T	25402717	25402717	KLF1	c.637T	12885593	12885593
RHCE	c.364C	25402718	25402718	KLF1	c.621G	12885609	12885609
RHCE	c.361T	25402721	25402721	KLF1	c.569del	12885661	12885661
RHCE	c.350_358del	25402724	25402732	KLF1	c.551_	12885674	12885679
RHCE	c.356A	25402726	25402726		556delinsA
RHCE	c.344C	25402738	25402738	KLF1	c.533A	12885697	12885697
RHCE	c.344G	25402738	25402738	KLF1	c.519_525dupC	12885699	12885716
RHCE	c.341A	25402741	25402741		GGCGCC
RHCE	c.340C	25402742	25402742	KLF1	c.519_520insC	12885709	12885712
RHCE	c.340T	25402742	25402742	KLF1	c.519G	12885711	12885711
RHCE	335 + 3t	25408680	25408680	KLF1	c.517del	12885713	12885713
RHCE	c.308T	25408710	25408710	KLF1	c.472del	12885758	12885758
RHCE	c.307C	25408711	25408711	KLF1	c.384insC	12885845	12885847
RHCE	c.307T	25408711	25408711	KLF1	c.380A	12885850	12885850
RHCE	c.286A	25408732	25408732	KLF1	c.310_311insG	12885918	12885920
RHCE	c.286G	25408732	25408732	KLF1	c.262_284dup	12885924	12885990
RHCE	c.254C	25408764	25408764	KLF1	c.304C	12885926	12885926
RHCE	c.254G	25408764	25408764	KLF1	c.298T	12885932	12885932
RHCE	c.221A	25408797	25408797	KLF1	c.204del	12886026	12886026
RHCE	c.208T	25408810	25408810	KLF1	c.196T	12886034	12886034
RHCE	c.203G	25408815	25408815	KLF1	c.151del	12886079	12886079
RHCE	c.202G	25408816	25408816	KLF1	c.114del	12886116	12886116
RHCE	c.201G	25408817	25408817	KLF1	c.109T	12886121	12886121
RHCE	c.187C	25408831	25408831	KLF1	c.90A	12886140	12886140
RHCE	c.178A	25408840	25408840	KLF1	c.86G	12887055	12887055
RHCE	c.150T	25408868	25408868	KLF1	promoter	12887479	12887479
RHCE	149 − 1a	25408870	25408870	GATA1	c.1240C	48794162	48794162
RHCE	148 + 5a	25420634	25420634	ACHE	c.1057C > A	100893176	100893176
RHCE	c.122A	25420665	25420665	ACHE	c.266G > A	100893967	100893967
RHCE	c.122G	25420665	25420665	ACHE	c.169G > A	100894064	100894064
RHCE	c.106A	25420681	25420681	ACHE	c.101G > A	100894132	100894132
RHCE	c.106G	25420681	25420681	ERMAP	c.169G > A	42830851	42830851
RHCE	c.105T	25420682	25420682	ERMAP	c.178C > G	42830860	42830860
RHCE	c.98C	25420689	25420689	ERMAP	c.139G > A	42830821	42830821
RHCE	c.93_94insT	25420693	25420694	ERMAP	c.242G > A	42830924	42830924
RHCE	c.94G	25420693	25420693	ERMAP	c.103G > A	42830785	42830785
RHCE	c.80_84del	25420703	25420707	ERMAP	c.307 308delGA	42830989	42830990
RHCE	c.84A	25420703	25420703	ERMAP	c.994C > T	42829761	42829761
RHCE	c.79_81del	25420706	25420708	ART4	c.144 + 2T > C	14842968	14842968
RHCE	c.48C	25420739	25420739	ART4	c.145 − 2A > G	14841155	14841155
RHCE	c.48G	25420739	25420739	ART4	c.185T > C	14841113	14841113
RHCE	c.28T	25420759	25420759	ART4	c.268C > T	14841030	14841030
RHCE	1 − 10t	25420796	25420796	ART4	c.323G > T	14840975	14840975
FUT1	c.1047C	48750235	48750235	ART4	c.343_350del	14840948	14840955
FUT1	c.1047G	48750235	48750235	ART4	c.350C > T	14840948	14840948
FUT1	c.991A	48750291	48750291	ART4	c.405C > A	14840893	14840893
FUT1	c.990del	48750292	48750292	ART4	c.431C > A	14840867	14840867
FUT1	c.980A	48750302	48750302	ART4	c.432C > A	14840866	14840866
FUT1	c.980C	48750302	48750302	ART4	c.442C > T	14840856	14840856
FUT1	c.958A	48750324	48750324	ART4	c.547T > G	14840749	14840749
FUT1	c.948C	48750334	48750334	ART4	c.566C > T	14840732	14840732
FUT1	c.948G	48750334	48750334	ART4	c.674T > A	14843243	14843243
FUT1	c.944C	48750338	48750338	ART4	c.793A > G	14840505	14840505
FUT1	c.944T	48750338	48750338	ART4	c.793A > G	14840505	14840505
FUT1	c.917T	48750365	48750365	ART4	c.793A > G	14840505	14840505
FUT1	c.903_	48750379	48750380	ART4	c.793A > G	14840505	14840505
	904insAAC			ART4	c.793A > G	14840505	14840505
FUT1	c.896C	48750386	48750386	AQP1	del all or part	30911853	30912293
FUT1	c.881_882del	48750400	48750401		exon 1
FUT1	c.882T	48750400	48750400	AQP1	c.112C > T	30912021	30912021
FUT1	c.881T	48750401	48750401	AQP1	c.113C > T	30912022	30912022
FUT1	c.880T	48750402	48750402	AQP1	c.134C > T	30912043	30912043
FUT1	c.832A	48750450	48750450	AQP1	c.140A > G	30912049	30912049
FUT1	c.826C	48750456	48750456	AQP1	c.232delG	30912141	30912141
FUT1	c.826T	48750456	48750456	AQP1	c.308_309insT	30912217	30912218
FUT1	c.801C	48750481	48750481	AQP1	c.576C > A	30922590	30922590
FUT1	c.801T	48750481	48750481	AQP1	c.601delG	30922614	30922614
FUT1	c.786C	48750496	48750496	ICAM4	c.299A > G	10287311	10287311
FUT1	c.785A	48750497	48750497	ICAM4	c.346_355del	10287358	10287367
FUT1	c.785G	48750497	48750497	ABCG2	c.2T > C	88139994	88139994
FUT1	c.776A	48750506	48750506	ABCG2	c.34G > A	88139962	88139962
FUT1	c.776T	48750506	48750506	ABCG2	c.34G > A	88139962	88139962
FUT1	c.768del	48750514	48750514	ABCG2	c.34G > A	88139962	88139962
FUT1	c.748T	48750534	48750534	ABCG2	187_197delATATTATCGAA	88139799	88139809
FUT1	c.725G	48750557	48750557	ABCG2	c.244_245insC	88132594	88132595
FUT1	c.725T	48750557	48750557	ABCG2	c.263 + 1G > A	88132575	88132575
FUT1	c.721C	48750561	48750561	ABCG2	c.289A > T	88131892	88131892
FUT1	c.695A	48750587	48750587	ABCG2	c.337C > T	88131844	88131844
FUT1	c.695G	48750587	48750587	ABCG2	c.376C > T	88131805	88131805
FUT1	c.694C	48750588	48750588	ABCG2	c.420_421insA	88131171	88131172
FUT1	c.694T	48750588	48750588	ABCG2	c.421C > A	88131171	88131171
FUT1	c.689C	48750593	48750593	ABCG2	c.421C > A	88131171	88131171
FUT1	c.684A	48750598	48750598	ABCG2	c.421C > A	88131171	88131171
FUT1	c.684G	48750598	48750598	ABCG2	c.421C > A	88131171	88131171
FUT1	c.682G	48750600	48750600	ABCG2	c.439C > T	88131153	88131153
FUT1	c.661T	48750621	48750621	ABCG2	c.440G > A	88131152	88131152
FUT1	c.659A	48750623	48750623	ABCG2	c.455T > C	88131137	88131137
FUT1	c.658T	48750624	48750624	ABCG2	c.458C > T	88131134	88131134
FUT1	c.655C	48750627	48750627	ABCG2	c.542_543insA	88121782	88121782
FUT1	c.649T	48750633	48750633	ABCG2	c.565_566del	88121758	88121759
FUT1	c.586C	48750696	48750696	ABCG2	c.706C > T	88118244	88118244
FUT1	c.586T	48750696	48750696	ABCG2	c.706C > T	88118244	88118244
FUT1	c.551_552del	48750730	48750731	ABCG2	c.730C > T	88118220	88118220
FUT1	c.548G	48750734	48750734	ABCG2	c.736C > T	88118214	88118214
FUT1	c.547A	48750735	48750735	ABCG2	c.784G > T	88118166	88118166
FUT1	c.545A	48750737	48750737	ABCG2	c.791_792delTT	88118158	88118159
FUT1	c.538C	48750744	48750744	ABCG2	c.875_878dupACTT	88115022	88115025
FUT1	c.538T	48750744	48750744	ABCG2	c.986_987delTA	88113510	88113511
FUT1	c.522A	48750760	48750760	ABCG2	c.1017_1019delCTC	88113478	88113480
FUT1	c.513C	48750769	48750769	ABCG2	c.1111_1112delAC	88113385	88113386
FUT1	c.513G	48750769	48750769	ABCG2	c.1384G > A	88099432	88099432
FUT1	c.491A	48750791	48750791	ABCG2	c.1515del	88097585	88097585
FUT1	c.462A	48750820	48750820	ABCG2	c.1515del	88097585	88097585
FUTI	c.462C	48750820	48750820	ABCG2	c.1591C > T	88097509	88097509
FUTI	c.461A	48750821	48750821	ABCG2	c.1714A > C	88095543	88095543
FUT1	c.461G	48750821	48750821	ABCG2	c.1723C > T	88095534	88095534
FUT1	c.460C	48750822	48750822	ABCG2	c.1789_1790insT	88094607	88094608
FUT1	c.442T	48750840	48750840	ABCG2	c.1819T > C	88094578	88094578
FUT1	c.424T	48750858	48750858	ABCG2	c.1819T > C	88094578	88094578
FUT1	c.423A	48750859	48750859	ABCG2	c.1820 + 1g > a	88094576	88094576
FUT1	c.422A	48750860	48750860	ABCG2	c.1841T > G	88092361	88092361
FUT1	c.422G	48750860	48750860	ABCG2	c.1858G > A	88092344	88092344
FUT1	c.371G	48750911	48750911	ABCG2	27-kb deletion	88165417	88165645
FUT1	c.349T	48750933	48750933	ABCG2	27-kb deletion	88164336	88164501
FUT1	c.328A	48750954	48750954	ABCG2	27-kb deletion	88164178	88164315
FUT1	c.293T	48750989	48750989	ABCG2	27-kb deletion	88158437	88158666
FUT1	c.269T	48751013	48751013	ABCG2	27-kb deletion	88158199	88158398
FUT1	c.235C	48751047	48751047	ABCG2	27-kb deletion	88157873	88158062
FUT1	c.35T	48751247	48751247	ABCG2	27-kb deletion	88156758	88156907
FUT2	c.4A	48702960	48702960	ABCG2	27-kb deletion	88150594	88150739
FUT2	c.40G	48702996	48702996	ABCG2	27-kb deletion	88145144	88145356
FUT2	c.113T	48703069	48703069	ABCG2	27-kb deletion	88139952	88140181
FUT2	c.244A	48703200	48703200	FY	c.125G	159205564	159205564
FUT2	c.244G	48703200	48703200	FY	c.265T	159205704	159205704
FUT2	c.278T	48703234	48703234	FY	c.298A	159205737	159205737
FUT2	c.302T	48703258	48703258	FY	c.680A	159206119	159206119
FUT2	c.379T	48703335	48703335	FY	c.125A	159205564	159205564
FUT2	c.385A	48703341	48703341	FY	c.145T	159205584	159205584
FUT2	c.385T	48703341	48703341	FY	c.266A	159205705	159205705
FUT2	c.400A	48703356	48703356	FY	c.901T	159206340	159206340
FUT2	c.412A	48703368	48703368	FY	c.281_295del	159205720	159205734
FUT2	c.428A	48703384	48703384	FY	c.408A	159205847	159205847
FUT2	c.481A	48703437	48703437	FY	c.287A	159205726	159205726
FUT2	c.569A	48703525	48703525	FY	c.327del	159205766	159205766
FUT2	c.571T	48703527	48703527	FY	c.395A	159205834	159205834
FUT2	c.628T	48703584	48703584	FY	c.719del	159206158	159206158
FUT2	c.658T	48703614	48703614	FY	−69C	159205371	159205371
FUT2	c.664T	48703620	48703620	FY	c.296	159205735	159205935
FUT2	c.665A	48703621	48703621		496delinsAGGCCACTG
FUT2	c.685_686del	48703641	48703642	FY	c.407A	159205846	159205846
FUT2	c.685A	48703641	48703641	FY	c.781A	159206220	159206220
FUT2	c.688_690del	48703644	48703646	FY	c.179_180del	159205618	159205619
FUT2	c.716A	48703672	48703672	FY	c.895A	159206334	159206334
FUT2	c.747_	48703703	48703704	FY	c.151del	159205590	159205590
	748insGTG			GYPA	c.250G	114118735	114118735
FUT2	c.778del	48703734	48703734	GYPA	c.250C	114118735	114118735
FUT2	c.818A	48703774	48703774	GYPA	c.244C	114118741	114118741
FUT2	c.849A	48703805	48703805	GYPA	c.244A	114118741	114118741
FUT2	c.853A	48703809	48703809	GYPA	c.242T	114118743	114118743
FUT2	c.868A	48703824	48703824	GYPA	c.242G	114118743	114118743
FUT2	c.950T	48703906	48703906	GYPA	c.240G	114118745	114118745
JK	c.130A	45730450	45730450	GYPA	c.240T	114118745	114118745
JK	c.838G	45739554	45739554	GYPA	c.239T	114118746	114118746
JK	c.511C	45736496	45736496	GYPA	c.239C	114118746	114118746
JK	c.28A	45730348	45730348	GYPA	c.232G	144119686	144119686
JK	c.226A	45731089	45731089	GYPA	c.232A	144119686	144119686
JK	c.742A	45739241	45739241	GYPA	GYP del exon 3	144119686	144119781
JK	c.838A	45739554	45739554	GYPA	c.230C	144119688	144119688
JK	c.548T	45736533	45736533	GYPA	c.230T	144119688	144119688
JK	c.718A	45739217	45739217	GYPA	c.226G	144119692	144119692
JK	c.202T	45731065	45731065	GYPA	c.226A	144119692	144119692
JK	c.582G	45736567	45736567	GYPA	c.217C	144119701	144119701
JK	c.956T	45748385	45748385	GYPA	c.217T	144119701	144119701
JK	c.561A	45736546	45736546	GYPA	c.212C	144119706	144119706
JK	342 − 1a	45734273	45734273	GYPA	c.203C	144119715	144119715
JK	c.723del	45739222	45739222	GYPA	c.197C	144119721	144119721
JK	c.866G	45739582	45739582	GYPA	c.197A	144119721	144119721
JK	c.27_50del	45730347	45730370	GYPA	c.191A	144119727	144119727
JK	811 + 5A	45739315	45739315	GYPA	c.164_166del	144119752	144119754
JK	342 − 1c	45734273	45734273	GYPA	c.160C	144119758	144119758
JK	c.222A	45731085	45731085	GYPA	c.148C	144119770	144119770
JK	c.499G	45736484	45736484	GYPA	c.148T	144119770	144119770
JK	663 + 1t	45736649	45736649	GYPA	c.140T	144119778	144119778
JK	c.871C	45739587	45739587	GYPA	c.140A	144119778	144119778
JK	c.896A	45739612	45739612	GYPA	c.140C	144119778	144119778
JK	c.191A	45731054	45731054	GYPA	c.138T	144119780	144119780
JK	c.194A	45731057	45731057	GYPA	c.138A	144119780	144119780
JK	c.512A	45736497	45736497	GYPA	c.107C	144120519	144120519
JK	c.437C	45734369	45734369	GYPA	c.107G	144120519	144120519
JK	c.536G	45736521	45736521	GYPA	c.72T	144120554	144120554
DI	c.2561T	44251253	44251253	GYPA	c.72G	144120554	144120554
DI	c.2561C	44251253	44251253	GYPA	c.71G	144120555	144120555
DI	c.1972A	44254581	44254581	GYPA	c.71A	144120555	144120555
DI	c.1972G	44254581	44254581	GYPA	c.68A	144120558	144120558
DI	c.1669A	44255804	44255804	GYPA	c.68C	144120558	144120558
DI	c.1669G	44255804	44255804	GYPA	c.67T	144120559	144120559
DI	c.1643T	44255830	44255830	GYPA	c.59C	144120567	144120567
DI	c.1643C	44255830	44255830	GYPA	c.59T	144120567	144120567
DI	c.1654T	44255819	44255819	GYPA	c.58T	144120568	144120568
DI	c.1654A	44255819	44255819	SLC35C1	c.439C	45806240	45806240
DI	c.1294T	44257796	44257796	SLC35C1	c.588G	45810828	45810828
DI	c.1294C	44257796	44257796	SLC35C1	c.923C	45811163	45811163
DI	c.1694C	44255779	44255779	SLC35C1	c.439T	45806240	45806240
DI	c.1694G	44255779	44255779	SLC35C1	c.923G	45811163	45811163
DI	c.1707A	44255766	44255766	SLC35C1	c.588del	45810828	45810828
DI	c.1707C	44255766	44255766
DI	c.1967A	44254586	44254586
DI	c.1967G	44254586	44254586
DI	c.1966T	44254587	44254587
DI	c.1966C	44254587	44254587
DI	c.1663C	44255810	44255810
DI	c.1663T	44255810	44255810
DI	c.1937A	44254616	44254616
DI	c.1936T	44254617	44254617
DI	c.1936C	44254617	44254617
DI	c.1937G	44254616	44254616
DI	c.1681T	44255792	44255792
DI	c.1681C	44255792	44255792
DI	c.1287T	44257803	44257803
DI	c.1681G	44255792	44255792
DI	c.1287A	44257803	44257803
DI	c.1696T	44255777	44255777
DI	c.1696C	44255777	44255777
DI	c.1696G	44255777	44255777
DI	c.1653C	44255820	44255820
DI	c.1653G	44255820	44255820
DI	c.1438A	44257538	44257538
DI	c.1438G	44257538	44257538
DI	c.1462A	44257514	44257514

To further illustrate the present disclosure, the primer set, the kit, and the method for genotyping multi-RBC blood group system based on NGS provided by the present disclosure are described in detail below in connection with examples, but these examples should not be construed as limiting the claimed scope of the present disclosure.

Example 1

S1. A genomic DNA was extracted separately from 5 whole blood samples (H07951D, H07949D, H07934D, H07936D, H07939D), 5 plasma samples (H07945D, H07942D, H07943D, H07948D, H07938D), and 4 paraffin tissue samples (H07929D, H07940D, H07941D, H07947D) using magnetic beads, the concentration and purity were measured, and then 10 mM Tris-HCl was added to dilute each sample to 200 ng/μL. The quality of each sample was tested using 1.5% agarose gel electrophoresis, and qualified samples were included in the group and labeled separately.

S2. The primer pairs of each pool in Table 2 were used to prepare 3 amplification systems, including: specific primer mix 1 μL (pool1, pool2, and pool3); enzyme mix Pxp-1st-mix 3 μL; genome template 1 μL (200 ng); nuclease-free water 7.5 μL, total volume 12.5 μL. There were 3 tubes of specific primer mixture in total, and 1 sample needed to be divided into 3 tubes for PCR.

A PCR reaction program of the first amplification included: initial denaturation at 98° C. for 20 min; 9 cycles of denaturation at 98° C. for 15 s, annealing at 62° C. for 20 min, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 5 min; and heat preservation at 10° C.

S3. The PCR amplification products (pre-library products) of each sample were purified using purification magnetic beads. The remaining primers, gDNA and dNTPs were removed from the PCR product, and the amplification products were recovered.

S4. The purified products were further amplified by PCR using universal sequencing adapters (N5XX and A7XX) carrying an index.

A reaction system of the PCR amplification included: enzyme mixture Pxp-2nd-mix 6.5 μL; pre-library product 4 μL; universal adapter N5XX 1 μL, universal adapter A7XX 1 μL, with a total volume of 12.5 μL. The same sample included 3 pre-library products, which were added to 3 tubes of PCR master mix containing the same universal adapter.

The PCR reaction program was preferably: initial denaturation at 98° C. for 2 min; 23 cycles of denaturation at 98° C. for 15 s, extension at 68° C. for 60 s, and extension at 72° C. for 60 s; extension at 72° C. for 10 min; and heat preservation at 4° C.

As shown in FIG. 2, the amplification products of 14 samples all showed clear and bright target bands in the range of 200 bp to 500 bp, which was consistent with the expected design. Moreover, there was an extremely low primer-dimer content, and the size of the primer-dimer was obviously different from that of the amplified target fragment. This indicated that the PCR amplification primers and detection methods could break through the limitation of traditional methods, thus producing a large number of primer dimers to enhance the primer capture efficiency.

Example 2

Using steps S1 to S4 in Example 1, primer detection sensitivity verification was conducted on the genomic samples extracted from 4 whole blood samples (H08215D, H07953D, H07997D, H07951D), 4 plasma samples (H07949D, H07934D, H07936D, H07945D), and 3 tissue samples (H07942D, H07943D, H07948D) that passed the quality inspection. The samples in each case had a starting concentration of 200 ng/μL, and were diluted in 5-fold, 10-fold, and 20-fold concentration gradients. After dilution, the samples had concentrations of 40 ng/μL, 20 ng/μL, and 10 ng/μL, and were labeled with the sample name and concentration, respectively.

As shown in FIG. 3, the amplification products of the 11 samples were mainly concentrated at 200 bp to 500 bp, which was consistent with the expected design. Moreover, there was an extremely low primer-dimer content, and the size of the primer-dimer was obviously different from that of amplified target fragment. All 11 samples had clear and bright target bands at different template usage levels, and the target band could still be successfully amplified even if the template content was as low as 10 ng. This indicated that the PCR amplification primer and detection method of the present disclosure could break through the limitations of traditional methods and improve a detection sensitivity.

Example 3

Using the same method as in Example 1, primer detection repeatability verification was conducted on the genome sample extracted from whole blood samples (H07939D, H07938D), plasma samples (H07929D, H07940D), and paraffin tissue samples (H07941D, H07947D) that passed the quality inspection. Each of the 6 samples was amplified by 2 different operators on 2 different laboratory platforms according to steps S2 to S4 in Example 1, with a sample injection volume of 1 μL. The 6 samples were amplified by different operators in different laboratories using primers specific for a causative gene of large vestibular aqueduct syndrome.

As shown in FIG. 4, a total of 12 amplification products from 6 samples were mainly concentrated at 200 bp to 500 bp and there were no other non-specific bands, which was consistent with the expected design. Moreover, there was an extremely low primer-dimer content, and the size of the primer-dimer was obviously different from that of amplified target fragment. There was no difference in the test results between different operators and different experimental platforms. This showed that the PCR amplification primer and detection method of the present disclosure had desirable repeatability.

Example 4

17-System Blood Group Genotyping and Rare Blood Group Detection Based on Targeted Capture NGS Technology

1. Experimental Material Sources:

From the blood samples of voluntary blood donors tested by the Shenzhen Blood Center Rare Blood Group Bank from 2018 to 2023, 20 different types of rare blood group samples were selected, namely: No. 1 and 2 H−, No. 3 RHnull, No. 4 and 5 D−−, No. 6 CCDEE, No. 7 S+s−, No. 8 S−s−, No. 9 Fy(a−b+), No. 10 Fy(a−b−), No. 11 Le(a+b−), No. 12 Jk(a−b−), No. 13 K+, No. 14 Jr(a−), No. 15 Di(a+b−), No. 16 Di(a−b−), No. 17 InLu, No. 18 p, No. 19 RHD mixed field of view, and No. 20 RHCcEe mixed field of view.

These samples covered four types that were detected by different detection technologies and could not be solved by traditional serology, PCR-SSP, and Sanger, but could only achieve clear results through this technology of the present disclosure:

• • (1) 13 cases of rare blood group samples detected through blood group serology experiments;
  • (2) 3 cases of rare blood group samples detected by PCR-SSP genotyping without commercially available serological detection reagents;
  • (3) 2 cases of rare blood group samples deduced by Sanger without clear results using PCR-SSP genotyping and without commercially available serological detection reagents; and
  • (4) 2 cases with abnormal blood group serology test results showing mixed fields of view and Sanger showing all double peaks and unable to interpret the results.
    2. Experimental Procedure:

(1) When collecting venous blood, a sample of 5 mL/(person) was retained in an EDTA-Na2 anticoagulant tube and stored at 4° C.

(2) Blood group serological test was conducted within 72 h for 31 key rare blood groups in 17 target blood group systems. Only 11 rare blood groups were suitable for serological technology, while the remaining 20 rare blood groups could not be detected by serological test since there were no commercially available antibody reagents or the detection principle was not applicable.

(3) Genomic DNA was extracted from the 20 samples within 72 h, and genotyping was conducted using PCR-SSP, Sanger, and the technology of the present disclosure in sequence.

3. Experimental Results:

(1) Blood type serological test results: among the 20 samples in Table 4, only 13 cases obtained clear blood group results through serological test, namely: No. 1 and 2 H−, No. 3 RHnull, No. 4 and 5 D−−, No. 6 CCDEE, No. 7 S+s−, No. 8 S−s−, No. 9 Fy(a−b+), No. 10 Fy(a−b−), No. 11 Le(a±b−), No. 12 Jk(a−b−), No. 13 K+. There were 2 cases that could not be interpreted since their serological results showed mixed fields of view, while the other 5 cases were not suitable for serological test.

TABLE 4
Serological identification results of 20 rare blood group specimens

Results

H

RH

MNS

Kidd

Diego

Duffy

Lewis

Kell

(rare

Sample

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

Anti-

blood

NO

H

D

C

c

E

e

S

s

Jka

Jkb

Dia

Fya

Fyb

Lea

Leb

K

k

group)

1

0

+

+

+

+

+

0

+

+

0

+

+

0

0

+

0

+

H−

2

0

+

+

+

+

+

+

+

+

+

+

+

0

0

+

0

+

H−

3

+

0

0

0

0

0

0

+

+

0

+

+

+

0

+

0

+

RHnull

4

+

+

0

0

0

0

0

+

+

+

0

+

+

0

+

0

+

D−−

5

+

+

0

0

0

0

+

+

0

+

+

+

+

0

+

0

+

D−−

6

+

+

+

0

+

0

0

+

+

+

0

+

0

0

+

0

+

CCDEE

7

+

+

+

+

+

+

+

0

+

+

+

+

0

0

+

0

+

S + s−

8

+

+

+

+

+

+

0

0

0

+

+

+

0

0

+

0

+

S − s−

9

+

+

+

+

+

+

0

+

0

+

+

0

+

0

+

0

+

Fy(a − b+)

10

+

+

+

+

+

+

+

+

+

0

+

0

0

0

+

0

+

Fy(a − b−)

11

+

+

+

+

+

+

0

+

+

+

+

+

0

±

0

0

+

Le(a + b−)

12

+

+

+

+

+

+

0

+

0

0

+

+

0

0

+

0

+

Jk(a − b−)

13

+

+

+

+

+

+

0

+

+

+

+

+

0

0

+

+

+

K+

14

+

+

+

+

+

+

+

+

+

+

0

+

0

0

+

0

+

Unknown

15

+

+

+

+

+

+

0

+

+

+

+

+

0

0

+

0

+

Unknown

16

+

+

+

+

+

+

0

+

+

+

0

+

0

0

+

0

+

Unknown

17

+

+

+

+

+

+

0

+

0

+

+

+

+

0

+

0

+

Unknown

18

+

+

+

+

+

+

0

+

0

+

+

+

0

0

+

0

+

Unknown

19

+

mf

+

+

+

+

0

+

+

0

+

+

+

0

+

0

+

Unknown

20

+

+

mf

mf

mf

mf

0

+

+

0

0

+

+

0

+

0

+

Unknown

(2) Genotyping results: three different genetic testing technologies were used to conduct genotyping of 20 rare blood group specimens. The results were detailed in Table 5.

In 7 samples, genetic testing technologies based on SNP sites, such as PCR-SSP and MassArray nucleic acid mass spectrometry, were able to confirm blood group results; for example, for rare blood group samples No. 14-16 without commercially available serological detection reagents, their rare blood group types obtained through PCR-SSP were: No. 14 Jr(a−), No. 15 was Di(a+b−), and No. 16 Di(a−b−). In the other 13 samples, due to complex and unclear genetic polymorphisms, the primers designed based on SNP sites were off-target and the genotype could not be determined.

Sanger was conducted to sequence the gene coding region, and clear allele types could be directly obtained for 10 pure and mutant samples. Another 7 cases had multiple heterozygous mutation sites, large fragment insertions, and hybrid recombination. In this case, Sanger could not directly differentiate between these different heterozygotes. Because Sanger generally produced mixed signals that were not sufficient to distinguish a relative proportion of multiple alleles, and predictions could only be made based on guesswork. The technology of the present disclosure could solve such problems more accurately. Through SNP detection and haplotype analysis, multiple alleles could be more effectively identified to provide more information to infer haplotypes (allele types). For the No. 17 and No. 18 of rare blood group samples deduced by Sanger without clear results using PCR-SSP genotyping and without commercially available serological detection reagents, their rare blood groups were speculated by Sanger to be: No. 17 InLu, No. 18 p.

There were also 2 cases No. 19 and No. 20 with abnormal blood group serology test results showing mixed fields of view and Sanger showing all double peaks and unable to interpret the results. The Sanger found dual genetic backgrounds that were indistinguishable. The technology of the present disclosure could obtain the blood group chimera allele types by analyzing the relative proportions of SNPs, which were: No. 19 RHD*01 (70%)/RHD*01N.01 (30%), No. 20 RHCE*Ce (20%)/RHCE*cE(80%).

TABLE 5
Comparison of genotyping results of 20 rare blood group specimens using three
different types of genetic testing technologies

	PCR-SSP and MassArray
	nucleic acid mass
	spectrometry (detection
	technology based on SNP

sites)

Sanger

Technology of the present disclosure

		Whether			Whether			Whether
		blood			blood			blood		Con-
		group			group			group		firmed	Was it
		could			could			could		rare	consistent
	Experi-	be	Analysis	Experi-	be		Experi-	be		blood	with
Sample	mental	con-	of	mental	con-	Analysis	mental	con-	Analysis	group	serology
NO	results	firmed?	reason	results	firmed?	of reason	results	firmed?	of reason	types	results?

1	c.881_	Yes	/	c.881_	Yes	/	c.881	Yes	/	H−	Yes
	882delTT			882delTT			882delTT
2	Ampli-	No	Complex	c.360-	Spe-	The	c.360-400del	Yes	Allelic	H−	Yes
	fication		gene	400del/c.	culated	sequencing	FUT1*01/		types
	failure		poly-	551_		result	FUT1*		could be
			morphism,	552delAG		was	01N.06		obtained
			primer			heterozygous			by
			off-			and			analyzing
			target			the allele			the
						type			relative
						could not			proportions
						be			of SNPs
						directly
						obtained
3	Ampli-	No	Whole	RHD +	Yes	/	RHD +	Yes	/	RHnull	Yes
	fication		gene	RhCE			RhCE
	failure		deletion,	deletion			deletion
			primer
			off-
			target
4	Ampli-	No	Whole	RHD	Yes	/	RHD	Yes	/	D−−	Yes
	fication		gene	deletion			deletion
	failure		deletion,
			primer
			off-
			target
5	Ampli-	No	Large	/	Spe-	Large	CE exons 3-	Yes	Allelic	D−−	Yes
	fication		fragment		culated	segment	9/CE exons		types
	failure		recomb-			heterozygous	2-7		could be
			ination,			recombination,			obtained
			primer			unable to			by
			off-			obtain			analyzing
			target			allele			the
						type			relative
									proportions
									of SNPs
6	Ampli-	No	Complex	c.48G/C,	Spe-	The	RHCE*CE/	Yes	Allelic	CCDEE	Yes
	fication		gene	c.150C/T,	culated	sequencing	RHCE*		types
	failure		poly-	c.178C/A,		result	CE.01		could be
			morphism,	c.201A/GA		was			obtained
			primer	c.203A/G,		multiple			by
			off-	c.307C/T,		heterozygotes			analyzing
			target	c.676G/C,		and			the
				c.722C/T		the allele			relative
						type			proportions
						could not			of SNPs
						be
						directly
						obtained
7	c.251GG	Yes	/	c.251C >	Yes	Conventional	c.251C > G in	Yes	/	S−	Yes
				G in		homozygous	GYPB*S/
				GYPB*S/		mutations,	c.251C > G in
				c.251C >		allelic	GYPB*S
				G in		types
				GYPB*S		could be
						obtained
8	Ampli-	No	Large	c.143C/T,	Spe-	There	GYP*Mur/	Yes	Serology	JENU	No
	fication		fragment	c.251GG,	culated	was a	GYP*Mur		test		(actually
	failure		inserted,	GYP(B1-		large			misjudgment		a
			primer	136-		insertion			discovered!		variant,
			off-	B?137-		before			It was		easily
			target	204-		the key			due to		missed)
				A205-		SNP site,			limitations
				229-		resulting			of the
				B230-		in frame			binding
				366)		shift, and			sites of
						it was a			commercially
						heterozygous			available
						mutation,			antibodies
						the allelic
						type
						could not
						be
						obtained
9	c.125AA	Yes	/	c.125AA	Yes	Conventional	FY*B/FY*B	Yes	/	Fy	Yes
						homozygous				(a − b+)
						mutations,
						allelic
						types
						could be
						obtained
10	Ampli-	No	Complex	c.287G >	Yes	Unconventional,	FY*01N.04/	Yes	/	Fy	Yes
	fication		gene	A/c.287G >		homozygous	FY*01N.04			(a − b+)
	failure		poly-	A		mutations
			morphism,			could
			primer			result in
			off-			allelic
			target			types
11	Ampli-	No	Complex	c.13G > A,	Spe-	The	FUT3*	Yes	Allelic	Le	No
	fication		gene	c.59T > G,	culated	sequencing	01N.01.05/		types	(a − b−)	(blood
	failure		poly-	c.202T > CA		result	FUT3*		could be		group
			morphism,	c.314C > TA		was	01N.03.03		obtained		antigens
			primer	c.484G > A.		multiple			by		were
			off-	c.508G > A		heterozygotes			analyzing		adsorbed
			target			and			the		and
						the allele			relative		difficult
						type			proportions		to
						could not			of SNPs		accurately
						be					identify)
						directly
						obtained
12	Ampli-	No	Complex	c.956C >	Spe-	Single-	JK*01N.04/	Yes	Allelic	Jk	Yes
	fication		gene	Tc.516	culated	base	JK*01N.12		types	(a − b−)
	failure		poly-	530del		mutations			could be
			morphism,			superimpose			obtained
			primer			fragment			by
			off-			deletions,			analyzing
			target			unable to			the
						directly			relative
						obtain			proportions
						allelic			of SNPs
						types
13	c.578TT	Yes	/	c.578TT	Yes	Conventional	KEL*01.01/	Yes	/	K+	Yes
						homozygous	KEL*01.01
						mutations,
						allelic
						types
						could be
						obtained
14	c.376TT	Yes	/	c.376TT	Yes	Conventional	ABCG2*	Yes	/	Jr(a−)	Serologically
						homozygous	01N.01/				unidentifiable
						mutations,	ABCG2*				(no
						allelic	01N.01				commercially
						types					available
						could be					antibodies)
						obtained
15	c.2561TT	Yes	/	c.2561TT	Yes	Conventional	DI*A/DI*A	Yes	/	Di	Serologically
						homozygous				(a + b−)	unidentifiable
						mutations,					(no
						allelic					commercially
						ypes					available
						could be					antibodies)
						obtained
16	c.1462AA	Yes	/	c.1462AA	Yes	Conventional	DI*02N.01/	Yes	/	Di	Serologically
						homozygous	DI*02N.01			(a − b−)	unidentifiable
						mutations,					(no
						allelic					commercially
						types					available
						could be					antibodies)
						obtained
17	Ampli-	No	Genetic	c.304T > C	Spe-	The	c. 1021T > C	Yes	Allelic	InLu	Yes
	fication		background	c.1021T > C	culated	sequencing	in KLF1*		types
	failure		different			result	BGM12/		could be
			from			was	KLF1*		obtained
			that of			multiple	BGM12		by
			Caucasian			heterozygotes			analyzing
			people,			and			the
			primer			the allele			relative
			off-			type			proportions
			target			could not			of SNPs
						be
						directly
						obtained
18	Ampli-	No	Complex	c.100G > A,	Spe-	There	c. 343A > T in	Yes	Allelic	p	Serologically
	fication		gene	c.343A > T,	culated	was a	A4GALT*		types		unidentifiable
	failure		poly-	c.418		short	P1.01/		could be		(no
			morphism,	428delins		insertion	c.100G > A in		obtained		commercially
			primer			before	A4GALT*		by		available
			off-			the key	02N.25		analyzing		antibodies)
			target			SNP site,			the
						resulting			relative
						in frame			proportions
						shift, it			of SNPs
						was
						superposition
						of
						multiple
						heterozygous
						mutations,
						the
						allelic
						type
						could not
						be
						directly
						obtained
19	Ambig-	No	Blood	Ambiguous	No	Blood	RHD*	Yes	Allelic	Chimera	Serologically
	uous		group			group	01(70%)/		types and		unidentifiable
			mosaicism			mosaicism	RHD*01-		proportions		(mixed
			led			led to	N.01(30%)		of two		fields
			to dual			dual			chimeric		of
			genetic			genetic			genes		view,
			back-			back-			could be		undeter-
			grounds			grounds			obtained		minable)
			that			that			by
			were			were			analyzing
			indis-			indis-			the
			tinguish-			tinguish-			relative
			able			able			proportions
									of SNPs
20	Ambig-	No	Blood	Ambiguous	No	Blood	RHCE*	Yes	Allelic	Chimera	Serologically
	uous		group			group	Ce(20%)/		types and		unidentifiable
			mosaicism			mosaicism	RHCE*		proportions		(mixed
			led			led to	cE(80%)		of two		fields
			to dual			dual			chimeric		of
			genetic			genetic			genes		view,
			back-			back-			could be		undeter-
			grounds			grounds			obtained		minable)
			that			that			by
			were			were			analyzing
			indis-			indis-			the
			tinguish-			tinguish-			relative
			able			able			proportions

									of SNPs

TABLE 6
Quality control results of sequencing 20 rare blood group specimens

Sample	Total	ReadMapped	ReadMap			Average
NO	Reads	Genome	Target	Uniformity	Specificity	depth	>=20x	>=30x

1	1123474	852942	784878	96.01%	92.01%	2845	97.62%	94.62%
2	1418628	891892	779782	90.97%	87.42%	2760	95.98%	92.98%
3	1075343	820595	748465	95.68%	91.20%	2636	96.34%	92.34%
4	1403989	1104238	954835	94.12%	86.46%	3404	97.76%	92.76%
5	1225134	881239	814157	88.73%	92.38%	2952	97.01%	92.01%
6	1325877	1027290	913159	90.56%	88.88%	3283	96.57%	93.57%
7	975678	638289	598483	85.56%	93.75%	2142	99.18%	96.18%
8	1478933	1045310	930013	96.63%	88.96%	3357	98.43%	95.43%
9	1373990	978968	910049	90.08%	92.95%	3255	95.89%	94.89%
10	925554	724617	672155	92.86%	92.71%	2425	96.74%	93.74%
11	819891	513006	449650	94.27%	87.63%	1578	98.91%	93.91%
12	1345798	866560	788917	87.42%	91.03%	2854	97.12%	96.12%
13	1190323	929881	901651	92.74%	96.95%	3267	99.45%	98.45%
14	1289037	793274	689141	95.39%	86.87%	2467	95.23%	94.23%
15	1432468	1063321	979000	97.72%	92.05%	3515	97.07%	95.07%
16	845192	674548	592861	89.16%	87.87%	2145	99.31%	96.31%
17	1420141	866002	799689	87.31%	92.33%	2872	97.83%	93.83%
18	1497523	1035088	939032	88.62%	90.70%	3325	98.27%	95.27%
19	804517	635247	595731	95.05%	93.77%	2155	98.82%	96.82%
20	1156893	796753	712979	94.28%	89.48%	2527	97.65%	94.65%

Although the above example has described the present disclosure in detail, it is only a part of, not all of, the examples of the present disclosure. Other examples may also be obtained by persons based on the example without creative efforts, and all of these examples shall fall within the protection scope of the present disclosure.