NL1044132B1

NL1044132B1 - Kit and method for abo blood group genotyping based on high-throughput sequencing

Info

Publication number: NL1044132B1
Application number: NL1044132A
Authority: NL
Inventors: Liang Yanlian; Peng Long; Wu Fan; Xu Yunping; Hong Wenxu; Su Yuqing; Liang Shuang
Original assignee: Shenzhen Blood Center Shenzhen Inst Of Transfusion Medicine
Priority date: 2020-08-31
Filing date: 2021-08-25
Publication date: 2023-04-19
Also published as: NL1044132A; CN112029842A; CN112029842B

Abstract

The invention discloses a method for ABO blood group genotyping based on high-throughput sequencing by capturing multiple PCR and a kit suitable for this method. Said method includes the following steps: Si. obtaining DNA samples of an individual subject; S2. carrying out PCR amplification to said DNA sample by a primer set consisting of primers having the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.248, respectively, capturing the target zone section, to obtain a DNA library; S3. further amplifying the DNA library by PCR amplification primers with sequencing adapters; S4. having the DNA library carry out high-throughput sequencing on Illumina platform to obtain sequencing data; S5. after pre-processing the sequencing data, carrying out ABO blood group genotyping according to the following sub steps. The objective of the invention is to obtain sequencing data covering 90.92% of the full length of the ABO gene, including all 7 exon zones, so as to more accurately complete the ABO blood group genotyping. 1044132

Description

ref: B 2021 NL 016

TITLE: KIT AND METHOD FOR ABO BLOOD GROUP GENOTYPING

BASED ON HIGH- THROUGHPUT SEQUENCING

TECHNICAL FIELD OF THE INVENTION

The invention belongs to the field of blood group genotyping, specially relates to a kit and method for ABO blood group genotyping based on high-throughput sequencing by capturing multiple PCR, 19

DESCRIPTION OF THE PRIOR ART

For ABO blood group in the blood group system having most clinical importance for humans, its incompatibility can cause acute hemolytic transfusion reaction and neonatal hemolytic disease, which can endanger patients’ life in severe cases.

Therefore, accurately identifying ABO blood group is the primary requirement to ensure the safety of clinical blood transfusion. However, ABO blood group will be affected by many factors such as diseases, subtypes, autoantibodies, irregular antibodies, and false or weak agglutination when detected by conventional serological methods, and sometimes cannot be accurately typed. in recent years, blood group genotyping technology based on the base sequence of blood group genes has bean established with the rapid development in genomics and the advance in concept of "precision medicine". At present, the ABO blood group genotyping technology quite commonly used in clinic is mainly the sequence-specific primer PCR {PCR-SSP} technology. Although molecular typing based on PCR-88P technology has high sensitivity and specificity, and can be used to identify known subtypes, it cannot be used to discover a new mutation,

Sequencing technology can accurately obtain the nucleotide sequence of a target gene segment and identify unknown mutations, so it is a golden standard for gene testing, enabling blood group analysis to reach a more refined level and greatly contributing to an assurance that the blood transfusion therapy is safe and effective,

Since 2011, high-throughput sequencing technology (also known as next-generation sequencing technology, NGS) has been first used in erythrocyte blood group genotyping, identifying the RhD blood group by sequencing the CDS zone. In the next few years, researchers begin to develop NGS typing technology for blood group genes. However, most of research on ABO gene sequencing mainly focus on the 6th and 7th exons, mostly analyzing the CDS zone, and rarely researching introns or upstream and downstream zones, one reason is that most of the mutation sites that affect protein function are located in the protein coding zone. However, the ABO gene is 24830hp in length, and the length of the 6th and 7th exons is only one-tenth of the length of the whole gene. Then, what role does the sections other than the two play in the expression of antigen? Some research on intron zones have confirmed that certain mutations in the intron zonss have an effect on the expression of ABO antigens. Only sequencing the 8th and 7th exons is not enough to guarantes the accuracy of ABO blood group genotyping. in addition, another important factor restricting ABO whole gene sequencing is that the GC content of Exons 1-5 of the ABO gene is extremely high, so it is difficult to design specific amplification primers, even if a primer can be designed, the amplification efficiency of the primer in these zones may also be very low. There is also research to capture the full-length ABO gene by probe capturing method, although itis free of primer design, the GC content or the amplification of low-complexity zons is still affected during amplifying the library. In addition, the lon torrent PGM sequencing platform has poor sequencing quality for the zone with high GC content and is not suitable Tor ABO gene test, which further restricts the development and application of

ABO whole gene sequencing. Until 2018, William J. Lane adopted the whole sxon- napluring sequencing method in the MedSeq Project to identify 38 erythrocyte antigens and 22 platelet antigens of 12 blood group systems, but this method has extreme cost, complex data analysis and long cycle, so itis extremely difficult to popularize if.

Compared with the above, the main advantage of gene-capluring sequencing is that it can sequence the specific zone, effectively reduces sequencing costs, increases sequencing depth, and has the ability to more accurately discover genetic variation information in the specific zone, so it is one of the foundations of precision medicine. A commonly-used gene capturing method includes hybrid capture and multiple PCR amplification. Among them, the characteristic of hybrid capture is that it can capture the target zone of the sxomes or even larger, but the operation process is complex and requires more specialized instruments and devices, while the multiple PCR capture is simple and flexible to operate, minimizes instrument requirements, can complste concentrating the target sequence and building the library within a few hours, and is suitable for capturing relatively small target sequence.

SUMMARY OF THE INVENTION

In order to solve at least one of the above technical problems, the invention realizes high-throughput sequencing to the whole ABO gene by setting up a multiple

PCR capturing sequencing process adapted to the lumina sequencing platform. On the one hand, # is used to screen the SNP that causes inconsistent forward and reverse typing of ABO blood, so as to further study its expression regulation mechanism; on the other hand, # is upgraded to the existing SSP and the first- generation sequencing technology, and establishes ABO blood typing system adopting the second-generation sequencing.

The first aspect of the invention provides a kit for ABO blood group genotyping based on high-throughput sequencing by capturing multiple PCR, the kit includes a primer set consisting of primers having the nucleotide sequences shown in SEQ ID

NOA-8EQ ID NO.248, respectively.

In some embodiments of the invention, the kit further includes an Index Barcode of a sequencing adapter, a multiple POR Master Mix, purified magnetic beads, and a positive control sample. in some embodiments of the invention, the kit further includes a DNA extraction reagent.

The second aspect of the invention provides a method for ABQ blood group genctyping based on high-throughput sequencing by caplring multiple PCR, comprising the following steps:

S51. obtaining DNA samples of an individual subject;

S2. carrying out PCR amplification to said DNA sample by a primer set consisting of primers having the nucleotide sequences shown in SEQ ID NO.1-SEQ ID

NO.248, respectively, capturing the target zone section, to obtain a DNA library;

S3. further amplifying the DNA library by POR amplification primers with sequencing adapters;

S4 having the DNA library carry out high-throughput sequencing on lllumina platform to obtain sequencing data; 85. after pre-processing the sequencing data, carrying out ABO blood group genotyping according to the following sub steps:

S51. comparing the sequence with the human reference genome by using a comparison software,

S552. analyzing the variation type existing in the sample, and adding annotation information to the variation site by using an annotation software,

S53. analyzing the comparison result and the annotation result, and obtaining the haplotype of the variation site of the sample, 854. comparing the haplotype result of the sample variation site with the ABO blood group system database to obtain the ABO blood group typing.

in some embodiments of the invention, the method further includes a step of purifying the DNA library with magnetic beads between SZ and S3.

In some embodiments of the invention, the method further includes a step of purifying the amplified DNA library by using two-step magnetic bead purification between S3 and S4.

In some embodiments of the invention, the method further includes a step of carrying out gsi electrophoresis quality inspection to the library before 83, if the main gel electrophoresis band is about 400bp, the quality inspection is qualified.

In some embodiments of the invention, the pre-processing in step 55 refers 10 performing quality inspection and filtering on the sequencing data, removing low-quality sequencing data, and processing adapter sequence data. in some specific embodiments of the invention, the data quality-controlling standard is as follows: 1} removing the reads containing an adaptor; 2) removing the reads whose N ratio is greater than 3%; 3} removing the low-quality reads (the hase number with quality value Q <= 3 accounts for more than 50% of the entire read),

In some embodiments of the invention, the comparison software is BWA software,

In some embodiments of the invention, the annotation software is annovar software, in some embodiments of the invention, the ABQ blood group system database includes ABO blood group data in dbRBC and ISBT databases, as well as other existing ABO blood group data. in some embodiments of the invention, if the ABO blood group genotyping result obtained in 854 is not unigue, the method further includes a step of analyzing the population frequency of the candidate typing of the ABO blood group to get the most probable typing result of the ABO blood group.

in the invention, the high-throughput sequencing is completed by one selected from high-throughput sequencing platforms including but not limited to Ilumina Miseg,

Xien, and Novased.

Compared with the prior art, the invention has the following beneficial technical affects:

An objective of the invention is to obtain sequencing data covering 80.92% of the full length of the ABO gene, including all 7 exon zones, so as to more accurately complete the ABO blood group genotyping. However, we find that the amplification- capturing analysis result is better than expected, with target zone coverage 298%, specificity 285%, uniformity 280%, and sensitivity 21% SNV by using the kit of the invention during actual testing.

Compared with the probe capture method, the method according to the invention has lower cost and relatively simple steps. 100% of (50/50) samples can be accurately typed on the three blood groups of

A, B, and O by adopting the invention, as shown in the subtype comparison results, 98% {49/50} of the sample subtyping results are completely consistent with the first generation sequencing results, while only one case of inconsistency is that the sample was a microchimera, so the first-generation sequencing fails to obtain a clear typing result.

A new variation site of ABQ can be found by adopting the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FK3,1 is a flow chart of primer design.

FIG.2 ts a flow chart of biological information analysis.

FIG.2 is a flow chart of ABO typing.

FIG,4 is an ISV visualization diagram of c.528C among the four characteristic sites of A101 in the sequencing result of Sample SN 16,

FIG.S is an IGV visualization diagram of c.703G among the four characteristic sites of A101 in the sequencing result of Sample SN.16.

FIG.8 is an IGV visualization diagram of c.796C among the four characteristic sites of A101 in the sequencing result of Sample SN.186.

FIG.7 is an IGV visualization diagram of ¢.803G among the four characteristic sites of A101 in the sequencing result of Sample SN. 16.

DETAILED DESCRIPTION OF THE INVENTION

In order to make the technical problem solved by the invention, the technical solution, and the bensficial effect clearer, the invention will be further described in detail as follows with reference to embodiments.

EXAMPLE

The following examples ars used hers fo demonstrate preferable smbodiments of the invention. A person skilled in the art will understands that the technology disclosed in the following examples represents the technology discovered by the inventor that can be used lo implement the invention, and therefore can be regarded as a preferable solution for implementing the invention. However, a person skilled in the art should understand according to this spacificgtion that many modifications can be made to the specific embodiments disclosed herein, and the same or similar results can still be obtained without departing from the spitit or scope of the invention.

The experimental methods presented in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all routine instruments and equipment in the laboratory. The test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent a companies.

Example: ABO Gene Sequencing Analysis 1. Material and Method 1.1 Sample Information 50 DNA samples in total have 30 cases of inconsistent forward and reverse typing of ABO samples {SN 1-30), and 20 cases of consistent forward and reverse typing of ABO samples {SN 31-60), all samples include forward typing, reverse typing, and resuits of PCR-SSP and Sanger sequencing.

Table 1 Sample information table

Original CT a | Sanger

Sample SN | typing result PCR:S5P i HOS142D | A102/A102 A AR AA | AA 2 HO6143D 002/001 0 | B 040, | 0,04 4 HOA1450 A | A{Ant- B”) AD: | Are

7 HO6148D | OM2/AI02 | A A{Anti-BY) AO i i 8 HO6149D | 00200 | © | B Uo0w i } 9 HO61S0D | A102/001 A | A(AmiB”") Arana

HOSISID | OGI/BI01 a | B BO, BO. i HO61S2D | O01/BA02 AB | B / | B{A)eDn i } i 12 HO6IS3D | OM/A102 A | A(Anti BY) AO; | AO: 3 | HOGISaD | 001/077 Cowon i ì i . en A101(6- | | ’ 8 < oe 1 i / i 16 HOGISTD | onion AeB AR AB 17 HO6158D | BA04/O91 | AB B BO, | B(Als/On ee: HO6I59D | A102/002 0 AOZ | Apf/On 19 HO6160D | O92/A102 0 LAD; | A 1202 i 20 HOGIGID | BI01/A102 AB AB | AroB oi

Lo HU6162D | BIOI/A101 AB 0 | AB Aro 1 b meeer nnn nen 1 1

C22 | HOGI63D | BIOV/A102 B AB LAB | AmB i i

Lon | HO6I64D | A102/002 A 0 AO; A 19202

Lag HO6165D | A102/A102 A oO | ; | A92 ee ee i | HO6166D | BIO1/A102 A AB | AB AroB io: 26 | HO6167D | A102/001 0 A LAD, At02001 i . . | | } : 28 | HOSI69D | OO1/B10! B | BO, BiOo1

2 | moan | owo0l | 0 | OgAniE™) at most72p | AI02/BI0I | AB | AB | AB x2 HOSIT3D AI03BIOL | AB | AB | AB | Ao a3 HOSIT4D AI03BIOL | AB | AB | AB | Awa 3 | HOSL7SD | ALOMBIOL | AB AB AB | AB | HO6176D | BIOL/ALDL | AB AB AB | ABe 37 | HOS17SD | 002/001 00; | OO 4 | H95186D | 002/A102 | A A a0, An 6 | H95187D | OOVW/BI01 | B } B Bo, | BO 47 HOSSSD | coumiot | B OBBO | BO 49 | HOSISOD | omit |B |B | BO, | meen O0UBIOI B |B | BO

Note: ”/ means that the bands are not aligned, and the result is not clear.

1.2 Primer Design

The capture target is the TuiHength zone of the ABO gene. We obtain the ABO gene locus information by searching the human ABQ gene at the ensembl website {chrD:136125788-136150617). The full length of the ABO is about 24,830bp, and the reference genome version is hg19. We carry out primer design by using the primer design process shown in FIG.1, After the Inventor examines, checks, adjusts and tests the primer, 124 pairs of primers are finally screened to cover 80.92% of the full length of the ABO gene, including all exon zones. See Table 2 for more detailed primer sequence information,

Table 2 primer sequence information en Size of amplicons

Amplicon Sequence (5-31) (bo) SEO ID Na, op; )

GCTOCTTGACTAGGGTGTCA

ABL I 218

CTGTGCCCAGATCACCCTG | 2

CAATGGCCCOAGACAGAGAC | 3

ABO_2 be 167 eeens

CGCTCCACCTTCGAGTTGT | 4

CAACTCGAAGGTGGAGCGG | $

ABO 3 | 267 jp

TGTCCTCCTUCCTTTTGGAA | 5 1

GCTTTGCTGAATTCCAAAAGOG 7

ABC 4 261 eee)

GTGOTGGTACCTCCTATCAC 8

ATAAGCCACCTTOTGCCAOG 9

ABO § — 263 en

ATTAGCCTTTAGOGTGCCTTGG | i

CTGGGGAAGGACCTGTGAGA | 1

ABO Bf 275

OCTGGGATTTTGCCATSGAGC

ATGGCTAAACATGGCTGAGC

I XJ J eee 263

GAGTGCAGTCGTGAAATOTGG

CAGTGAGCCCACATTTCACG

ABD B 270

CAGCTGGGGCATCATCAGTT

TTCCTGCTCAGATCCTGGOT 17

ARO 9 meeneem 261

TTTGCTTECTTGCCCCTETC 1%

GAGGAAGCGGGTGOATCAAG 19

ABO_10 EE 269

GTTCAAGTCCAGGCTGACTCC 20

AGATGACACTTTGGGGCTGC a

ABO_11 nnen 257

GGGGATTTCCAGGOOTTGTC 22 i

CCAAGCAATTTAGGCGCCTG | 23

ABO IZ bree 267

CAAGGGAGGGGAAGGAGCA | 24

GGACACAGOTCAACTCCAGT | 25

ABO_13 een 264

TCCTGACCGTTGTACAGCAG Las

CTACTTCAGAGCTGCCAGGC | 27

ABO 14 fee 274

AGCTTCTTATOTGCCCCACC | 28

AGGTOTGTOCTCGATTTGGG | 29

ABO IS freee 272 TT

ACTGTCCTTAGTGTAGGGCO | | 30

TCCACTTCCAAGCAAGGTAGA | C3

ABO_I6 | 275 feeen

TTGOTGTTTCOTGTTGCTaG | |;

TAGGGACCTTOGGGACCTGAG 33

ABO_17 —— 158 ie

TGAGGGCCAATTTOTTGGG 34 i

CAATAAGTTGGCCCTCAGC | 35

ABO_18 ee 238

TTGTTTTCTTGATTTCTTTOTTAGGGT | 36 1 meee

CCCTAAAGATTCCACCAATAAGATGT 37

ABO 19 eeen 27} i 3

TTGGCACCTGATCTCACATATGG

AGGAAGTGAAAGATCTGTATGUTGA | 39

ABO 20 ee 274

GGAGCAGGTGTTACTCCCTC 40

TGTCCATACAACCCCAAACAATC

ABO _21 271

ATTTAGGGGTCTTTGTGGCCA

AGAGOCCTAGGAGACATGAG | 43

ABO 12 275 i

TCCACATTGTGTTTCTTGGCA | 44

GAATAGAGAGCCCGGAAGTGA | | 45

ABO 23 256 —

CCACGOTAAGGTCCTGAGGA 46

AGACCTCCTCCATGATCCUT | 47

ABO 24 272 —

TGCATAGAGACACATGGAGAC | 48

TTGGGCTGCCTAAGTCTGTG

1

ABO_25

ACACCAGACACGUAAAGACA 50 : _

TGTCTTTGCGTGTCTGGTGT si

ABO 26 160 -

AGAAAGACACACATAGETCCC $2

CGGGAOGOCOGTATCTGTGAT 53

ARQ 27 rr 240 me

GACACACAATCACACGCAGC 54

TGTGTTTOTGTGOCCGOAA SS

ABO 28 frst 278 ee

GTAACTDAAGCETAGGCCCD | $6 b 1 ! 1

GOGGTOTGTOTGATTTGAGG | 57

CCGAGTACTTGTGGUACCAG

GAACCTCAGCTTCCTCAGGA | 59

ABD 30 253

GAACCTCAGCTTCCTCAGGA | 60

GTGGATTACCTGGTGTGCGT 61

ABD 31 264

OTCGACCATTATGGCCTGG | 62

TGAAGCTOTTCCTGGAGACG | 63

ABO 32 266 A

GTCGCGGAACTCCATGTCC 64

CUATCGCTGGGAAGAGGATG | 6

ABO_33 rns 246

GCCCACCATGAAGTGCTTCT 66 ee

GCCTCGCGTOCAGCTTATA 67

ABO 34 ee 262

AGATGCACCACGTTCTCCTG 68

ACACCAAAGGAAGTGGCTGA 69

ABO_35 mee 275

COCCTCTTATGGCCAGGC 70

CCTCTCTGTAACTGTGGCCG no

BBO TIG rrr 263 ee

TAGCTCCCTCTCTGGCCTG 72 eee in

GCAGGAAACATCTGUAGCCT 73

ABO IT jen 259 —

TGTOTTCAAGCACAGAGTGGOO 74

GCTGCATGAATGACCTTTCCC 75

ABO IE en 273 mee

ATGTCCTCOTGSTACCCCTT 76

ATGTGCCOTCCCAGACAATG 7

ABO 39 nn 253 —

TOOGAATOATTTGCCCGOTT 78

AACTCCACTCAGCTTCTGCC 9

ABO 40 261 rnd

TTOTTCTCCAAAGCCCCTGC EO

AACAAACCCTCCCCAGCAC 8

ABO_41 ee 262

GCTCAAGGGGUTGTTCTGAA 82

CATCAAGGAAACCGCCCTCT 83

ABO_42 een 267 9

COCCAAGCAACCACAGTOAT 84

GAAAGTOTGGGATGCAGGTA 8s

ABO_43 een 270

GAAAGCGTGGGATGCAGGTA 86

TGCGTGTGACACTTGACAGT | 87

ABO_44 ee 372 ee }

GGCCATTGCTTTCCACTTGA | 88

CTCACGGTCCATCCAGGTTO 89

ABO 45 eene 271 rrr

CCGGOTTCCTOTCTGAGAAG 90 eee eee eee

TTGCCAACCTGAAGCTCTGT | 9

ABO 46 fe 234 I Ah PH.

AGCAGGOCAOTCAGCAGAAAA | 92

SGGCTGTTTCCCAGGATGTG | 93

ABO 47 eee] 265 ems

GGOAGGGAAAATGGAGTTGC | 84

OCACOTGGCCTTCAAATTGG | 98

ABO_48 eee 122 me

GGCCTTGATTGCCTAGGTCA | 96 k

GTCAGTOOCTCCCTTCAGTS | 97

ABO 49 ee ) 251 ee

CACACACACACAAAAGCCGG | 98 i

GOCTGCAGGACAATTCTATG 99

ABO 50 ee 271 ee]

ATCGCCACAGTGATGOTTOT 100 $

CAGAGGCCACCAGAAACAGT

ABO 31

AGCOGTCACTTICTGCTGCAT

TCTACAAACCATGAGGUCCC | 103

ABO 52 267

TGCAAAGACTTCCCTGCATCT L104

TOTCTCAGATGACAGCCAGC | 165

ABO 53 275

CTATGGCCTCTGTTGOGACT 106 $ ==

TTAGCAGACACAGCAGGGGA | 107 t

ABO 54 frst 275 eenn

GACTTGTCTCCCCATCCCCA | 108

CAGGAAGGCAGGATCAGAACA | 109

ABQ _§§ eenen 271 on]

CAGCCATCCTCAACTCTCCC | 110

SE UU SU EE i

GGCTOGCAGGATTTGGGAA | 111

FN £7 JT J 238 me

GGCTGGCAGGATTTOGGAA 112

:

GACCAACAGGCAGTCTTCGT uso

ABO_37 | 267

CCCTTCAAGGCCACAGAGTT 14

GGTGCCCATACGTGAGCAAT | BERET:

ABO 58 | | 270

TGGCATTAGACTTCTGGGGC | Cus i i

GTCCCAAGCTGTTAGTCGCT | Co - 1 i

ABQ_39 | 275

TTCTCAGCAAAGCTCAGGCG | Ls

AGGACAGTTATTGCOTGCGT

ABO 60 1- 264

CCCCAGTAGAGGTGACAGGT

AAACCAAAATGCCACGCACT

ABO_61 235

AGCAAGTAAGCACACCUTCE 122

TGGGTTTGGAGATGCTCAGG 123

ABO 62 | 273 ed

AGCCTCGATGGTCAAATGTGT 124

CTGCCAAGATGGTCCCTCTG 125

JE LY eene 264 eee

GTGOGTTCGACAGTGACCTC L126 i | !

GOAGACCGGAATGAACCACA | TY

ABO 64 eee 271 tee

AACTGAAACTCCAGCAAGCG | DE

TCGCTTGCTGGAGTITCAGT | 129

ABO 65 263

CAGCCTCCTGGGACCAGA | 130

GCACCTTCOGCTCACTACA

ABO_66

TAAATTAGGCACCGGGTGGG

TGOTTGTTAATCCCACCCGG

ABO_67 242

TGAAAGGGACTGATTTCOGGG

TOTCCTGGCCCAGTTCCTAT

ABO_68 255

TCCACCAAGATTTCCCTACAGC 136 iS

CGAAACOTGGGAGGCAAAAC | CBT

ABO 69 proms 272 Fe

CGAAACOTGOGAGGCAAAAC | | 138 ed

TTGTGOCGGATTAACAATGOC | 9

ABO_T70 freee 266 ed

TGTGCCCACTTTAGGTATACGG | I

ACCCATCTTTTCCATTCCTTGGTA | 141

ABO_71 255 -

CACACCGCATGCTGTTITGA 142

ACCACTCCTCATCCACTAGCA 143

ABC 72 257 rr

GOCCACTAGUAACAAAGOTT 144

GAGCCATGATCATGCTAUCA 145

ARO 73 | 272 ee

AACCACAGTCCTTGCCAACA 146

AGCTAGATGTUGTOCATGTC 147

ABO 74 | 141 rrr

CACAGTCCCACTCCAGTTGC 148 ! i

AAGCTCAGGAGTTCGAGGTT 149

ABG 75 eee 256 —

AGGCATOCACCACATCTAGC 150

GACACTGGGTCTCATTAAGTTGTT 151

ABO 76 me 256

TCCCTACATACCCTCTCACCC 152

TGGTTCGCAGAGACTTGGAA

ABO TJ mee 181

CTGTGCCCGACCTCTCTITT

TGCTCAAGAGGATGGATACCAG

ABO Tg es 270

AAAGGCCCAAACCATAAAAATGG

TCAGTCGGTGGCTTATPTATICAT

ABO 79 L- 249

TCCATTGAGGGAAGAAAGGGAC 158

TGCCTACCCCAAGTTCATGA 159

ABO_R0 175 Ee

TGATAAGGGTGCTAAGGTAATGTGA 160

CAGTCATTTCAGCCATTTGAATTGA 161

ABO 81 me 275 me

TCCCCAGATGGATCTACAGAGT 162

CTGGGGTTTTAAGTGAGATTATGTGG 163

ABO S2 bs 238 ee

CTGGGGTTTTAAGTGAGATTATGTGG 164

ATATAAACCAGGCACCATOOC | L188

ABO_83 | 244 ef

AGGCTOGAATGCACTGCAAT | | 166

TTGAGTGTGOTGGTATGOCT | | UY A

ABO 84 | 227

TCATGGTTCAATTGATTGTGGCA 168

CTGTGAGCCCATCAGCTAAGT 169

ABO_85 227

CAGGTATACCACCACACTCA

TCAATTCAGTAAGACAAGTAAGOAGA

A

ABO 86 | 275

CAGCCTGATTTATTCATTGACTTTGT i

ACAGGAACTTGACTCCAGTATAACT

ABO 87 275

AGCAAGGAAAGGTCACCAGA 7!

Lo en

AACCACAAGCAAGGAGOGAG | 175

ABOLSB ee 237 —

TOTTCTTCTGTTCATCTTTTACTACCT | CTs i

TGGAGCAAACACTAACAAAATAACTT | | 177

ABO_89 eee 178 Jone

GGCAGCATATAGTCAAAGCTTAAAA 178

GGCAGAGATCGTTAGACTGGA 179

ABO_90 EN 151 mre

CTCCTCAAGCTCCCTTACGG 180 i

GCACACACCATCATGGCTG 181

ABD 91 boomers 275 ee

TGGGGCTAGGTTGTCAGGTA 182

CACCCCATCTGGCCAACTT

ABO_92 235

AGGACTCCAGCCCTTICTET

GCCCTAAAATAUTAGAACAGGA

ABOE ee 181

TCTTCOCAATGOCTGGGAAA | 186

AGGCATTGCGAAGACTCTGT | L187

ARO_94 245 el

AGGCATTGCGAAGACTCTGT ee

GCCCAACCOTTACTGGTGAA

ABO_95 235

CCCCTGCACAAATCTCOCT

COTCCCAAGGUTTAGGTCAC

ABO 96 7 275

CCCOCAGCAATACATGAAGC i

CTCTTGOCAGGTGGAGTGTT

ABO 97 +- | 274

CAGGAGGCTGAGGTGACCTA | 194

GATTTTCTTCTTTGACCCATTGCTT | | 195

ABO 98 bee 275 — ; }

TCACTGGACACCAACTGACA | L196

ACTTACAATGGAAGCTTACACACT | 97

ABO 99 eee 27 ri

TCTTCTCTAGCCACAGGGGA 198

AAGATTGGAATTGCTGTCTCCTCT 199

ABO 00 eee 270 en

TCAAGTGTGTAAGCTTCCATTGT 200

TGACCAGAGAGAGAGAGAACACA 201

ABO_101 | 229 me

TCTAAGAAATCTTAATCCAGTOTCACA 202

ACACATAAACTAGAAAGCGGTTAGAA 208

G 7

ABO 102 ’ | 274

CAGTTTTGAGAGTTCTTTACATACACA

GTAAAACTTTTCTACTTCAAAACAUCA | 205

ABQ _103 264 _

CCCCAAGGACAAATGATGGC | | 206 me eee

AGGCAAAATATTTCAACAGGC | | 207

ABO_104 mee 183 ed

GCTGAGGGTTCCACTGTCTG | 208

AGCCTGGATGTOOTGGTACTC 209

EE [rmmssmmmmmemssstsmmissmstiosmmtssssomiiios 227 ee

TTTCCAAAGTGACTGOCCCCA 210

AATGGGGCAGTCACTTTGGA | 211

ABO 106 bee 97 ee]

COTTGATACCCAGCAGAGGA L212 b 3

TOCTETGCTGGGTATCAACC 23

ABD HT frites 275 st

TOOTCTTTTGCATCTGACTTCC | 214

ACAACATACATGAATCTCGGAATCA 215

ABQ HI fromm 206

TAACTTTTCCATCCCCAGCCC 216

GACTGCAGAGGAGTTCCAGG

I ABQ 105 eee 273 i

TOTTACTCGGAATGCTOCCC

GOTOTGTTCAGTAGGGCTCC b 3

ABO_110 239

TTGTCATGCTTGAAGGCCCA

TGATTTAGGAGACAAGATAAGGOTIT

ABO 111 278

ATGCAGATGAACTGGGGAGA

CCAGGTCTTCTCUTTGTAACTGG

ABO 112 262

ATGCTCAGOTCOTTCACACT 224

AAAACTCCTGUACGTGGTCT 5 225

ABO 113 - 271 pee

AAGCATGCCAGGGAGAAGAG L226

CCCCAACAAGCCTGACTGAT Cor

ABO 114 ee 265 pe

AGAAACCTOGCAAATGAGTGC [228 i

TTCTAGCACAOGCCTGCTTG | 229

ABO TIE eee 273 ee]

COCCACGGSTTATGCTGTC 230

CCAAGGCTCCCTGACATGAG 231

ABQ IIG frm 267

CTGGGACTCAGGTCTGACTG 232

GACCACAAAGGAGUGATAGG 233

ARQ 117 272 : i i

CCTTGCCCTCAGTAATGUTC | 234

TTGAACACAGTGTTGCCTCA | 235

ABO 118 eee] 275

TACCCAGAGCCAGCACALCA 236

CTCACTAAGTCAGGUCTCAG

ABO 119 Fr

TITCTTGGUGCCAGAAGATO

TOTGCATCCCTCOCTCTGAA

ABO 120 eme 275

GAACAGCCTCCATATCCCAA 240

OCATTGAACTTGCCCACCTE | 241

ABO 121 bne] 225

CGGTCTCCASTCTCTCCT | 242

ATAGCAGCTCATGGAAGGCG | | 243

ABO 122 feeen 257

CTCAGUGCTGCGAGOCTT | 244

CAGGGTETCGGAGGCTGG 245

ABO 123 eeen 197

GGAGOCCGAGACCAGACT 246 eee feeen

GAGOCCTGCACTCACCG 247

ABO_124 ete 227 eee

GCCOTCCCTTCCTAGCAG 248 1,3 NGS sequencing 1.3.1. Setting up a sequencing library (panel: ABOv2.0}

PCR amplification: amplifying the target zone section through specific primers $ to concentrate the target zone section.

Magnetic bead purification: removing the non-amplified section by the magnetic bead purification method, and improving the purity of the amplified section in the target zone.

Library amplification: amplifying the DNA library in multiploidy by PCR amplification primers with sequencing adapters, so that the library concentration meets the sequencing requirements.

Library purification; sorting and purifying the amplified DNA library by using two- step magnetic bead purification.

Computer sequencing: sequencing on the computer (lumina sequencing platform) to obtain sequencing data after the purified DNA library has been qualified and guantified. 1.3.2 Quality inspection standards

After the library is amplified, we have to carry out gel electrophoresis quality inspection to the library, and the main gel electrophoresis band is about 400bp. 1.4 Sequencing data analysis

We carry out quality inspection and mutation analysis to the sequencing data by a biological information analysis software, The analysis flow chart is shown in FIG, 2.

The specific analysis includes the following steps:

Original data quality inspection: testing the quality of original sequencing data.

Data filtering: filtering low-quality sequencing data, processing adapter sequence data.

Comparation: comparing the sequence with the human reference genome.

Capture analysis: analyzing the uniformity, specificity, coverage, sequencing 15 depth, etc, of the data.

Variation analysis: analyzing the indel, snv and other types of variation in the sample by using a variation analysis software.

Mutation result annotation: adding annotation information to the mutation site by using an annotation schware.

ABQ blood group typing analysis: obtaining the ABQ blood typing results of the sample by using the typing program "ABO _Typer”.

Quality inspection standards: raw data Q20>80%, Q30>80%, Unformity>90%, specificity>90%, coverage>80%, sequencing depth>1000x,

1.5 Genotyping

The genotyping module in the analysis process uses the program

ABQ _Typer.py to carry out typing analysis to ABO genes.

The program takes the file annotated by the annovar software and the comparison result file of the BWA software as an input file to obtain the haplotype result of the variation sites of the sample by analyzing the information of the two files raspectively(due to too long distance between the amplicons in the genome of some sites, no clear haplotype results can be obtained, so the program will automatically output all possible haplotype results of the candidates), the haplotype analysis results of the variation sites are compared and analyzed with the ABQ blood group system database {the database contents are mainly gatherad at dbRBC, ISBT databases, and a small amount are gathered at literature data} to obtain the candidate ABO gene haplotype results. There may be one or more pairs of haplotype results in this part of the results. Due to the short reads of the second-generation sequencing, itis impossible to directly and accurately obtain an unique haplotype result. The program analyzes the population frequency of candidate typing to get the most probable typing results, and finally output a pair of unique haplotype resulis{due ta the limitation of the database or because the frequency of the candidate blood group population is the same or close, typing cannot be done, in this case, the program will only output the candidate typing result, and manual verification is required). The flowchart of ABO genotyping is shown in FIG.3. 2. Results 2.1 Capture analysis result

We count the coverage of the sequence in each zone by comparing the sequence with the human reference genome (hg18 version), and finally obtain the coverage and uniformity of the target zone, meanwhile also count the specificity of the primer ampiified section, the effectiveness of data, etc. The analysis results from 3¢ amplifying and capturing 50 samples are good, the average sequencing depth is 4667 x, the sequencing depth of any sample is much greater than 1000x, the average specificity and uniformity are both greater than 80%, and coverage with >20x is greater than 98%, which can meet ABO typing requirements.

The results of the amplicon capture analysis are shown in Table 3.

Table 3 Amplicon capture analysis results 1 SN SN dGenome Target | tv {%) y {5%} | depth 0%} {%o)

} HOGIAZD | 1606060 876214 | 852369 96.77 | 94,35 1 2 | HO6143D | 843819 588495 | 524134 89.03 99.19 | 98.39 31 HOAI44D | 923017 | 677123 621415 94,35 | 91,73 5011 99,19 99.19 fre | ee 4 HOSI4SD | 903283 | 613852 94,35 | 42.9 4598 | 99.19 | 99.19

Le LL

HO6I46D | 1000000 | 674999 515350 | 96.97 91,03 4963 100 $9.10 i !

CTT

6 HO6147 | 734089 489778 425768 | 94.35 86.89 | 2433 | 190 100 7 Hool1asn | 746162 485346 | 415123 | vale 85.49 | 3347 | 100 83.39 8 HO61490 | 1000600 732569 | 677757 42.44 | 5465 99.19 | 9839 i i i 9 | HO6I50D | 905669 £57484 | 390432 | 94,35 90.45 4761 9919 | 9918 | HD6ISID | 365386 123104 264811 | 94.35 | 819 2138 | 9839 | 97.58 u BOG 924827 | 697589 630419 | 9032 | GLA! s1s6 | 8677 | 95.18 12 | MOGISID | 906973 | 674794 633690 | 9355 | 93,88 S110 99.1% | 98.39 {1a | Hosis4D | 865117 639049 573646 | 93.55 £9.71 4636 | 9019 | 96.77 14 | HOSISSD | 1000000 733802 702763 | 95.97 95.67 S667 | 100 i i

HOGIS6D | 839785 SG4817 | 540728 | 95.16 95.68 | 4360 | 9839 | 97.58 16 | HOGISTID | 1000000 606434 | 633009 87.9 908 | S104 | 9839 | 9597 17 | HOGISSD | 1000900 936769 922203 | 89.52 98.46 7447 100 | 9839 i 8 | HOS159D | 1090900 | 825315 804288 | 90,37 | 97.4 6486 | 99.19 | 99.19

Lo H06160D | 958057 | 742722 93.55 | 95.12 5699 100 160 i i i i mm fn mm mm me mm mn 26 | HOSISID | 627957 382353 356247 | 91.13 | 93.11 96.77 | 95.16 2 hosp | swsso | Tt | esas | so wa 22 | HOS163D | 1000000 931676 918813 | 80.65 98,59 | 1400 | 96.77 | 93.35 23 | HOGI64D | 839624 633434 | SR7264 | 93.88 9268 | 4736 | 100 98,39 rT | i : 24 | ween 719556 483252 | 414423 | 88.7 85,34 | 1342 | 97.58 | 92.74

| HOGIG6D | 806676 595850 553547 | 88.71 | 92.85 4464 | 97.58 | 96.77 ~ 26 | HO6I67D | 1090009 649749 | 95.97 91.33 | 5239 100 | 100 i 027 oeren | 703558 463236 403678 | 94.35 87.11 | 3255 28 | HO6169D | 991671 735199 | GBRD4G | 94.33 9363 | 5555 | 99.19 | 98.39 rm 29 | HOG6170D | 836734 621607 | 570450 4600 | 99.19 | 98.39 ° | HOG17ID | 1000000 | 652572 | 603891 | 95.97 $9.48 4870 | 100 99.19 } i i i : 31 | HQ61I72D | 839394 | 551259 515778 | 91.13 93.5 4159 | 97.58 | 95,97 32 | HO6173D | 756056 | 319972 480445 | 90,32 43,24 3874 | VEV | 9597

Cn mse | sie | cme | ses 35.16

U HOOITSD | 714116 479549 446526 | 88.71 | 43.06 9435 3s HOGIT6D | 685415 395824 | SETI | BRS 98.16 | 9274 i i i 26 | HOBITTD | 1000000 808417 THOS TO | 95.16 | 95.16 97.58 | 97.58 37 | HOGITED | §R4720 614601 | 573067 | 95.16 93.18 | 4621 97.58 | 97.58 38 | HOGI79D | 909274 624574 | 556204 | 95.16 89.48 | 4510 | 9758 | 97.58 39 | HO6ISOD | 831949 564778 | SI7086 | 95.16 91.5 | 470 | 9758 | 97.58 30 | HOGIRID | 887624 | 628350 | 600372 | 95.16 95.5 | 4841 | 975% | 97.58 41 | HOGIR2IY | 78321 | 507415 458086 | 93.53 90.2} 1694 | 97.58 | 97.58 i i 42 HOBI8 1 883657 | 51403% 479946 86.23 93,32 3870 | 95.16 91.94 43 HO61841s GS 1RE7 F74488 742173 93.55 95.7% 4985 | 97.58 97.58 ì i 44 | HOGISSD | 847078 612509 | 9435 94.54 4939 97.58 | HOGISGD | 701258 462041 423104 | MAS | 9153 3412 | 97,88 | 97.58 46 | HO6ISTD | 691805 456740 424314 | 9274 | 92.84 3421 97.58 | 97.58 :

HOGISED | 750176 530674 490262 | 9435 | 94.04 97.58 | 97.58

HOGISOD | 778248 575402 543338 | 9435 | 94.38 4381 i

49 | HO6I90D | 909671 | 706092 | 688559 | 94.35 97,48 50 | HO6191D | 787988 | 600414 | 569992 | 92,74 | 94,89

Aven | 85912} | 622038 | 578846 | 92.5954 | 92.4766 2.2 Comparison of identification resulls in this example, 50 DNA samples in total have been all taken into traditional serological testing (forward typing, reverse typing), PCR-SSP, and Sanger sequencing testing before the next-generation sequencing typing. In the three blood groups of A,

B, and O, the comparison results show that 100% (50/50) of the samples can be accurately typed. As for more detailed subtype comparison results, 98% (49/50) of the sample subtype typing results are completely consistent with the first-generation sequencing results. The specific comparison results are shown in Table 1. 2.3 Analysis of inconsistent results

As shown in the comparison results in Table 4, there is only one sample {Sample SN. 18) that first-generation sequencing fails to obtain a clear typing result, is but we find that the mutation abundances of four sites of ¢.526C, ¢.703G, ¢.796C, and £.803G of the sample related to A101 In the second-generation sequencing are all between 6 and 7% {F1G.4-7), which is suspected tc be a microchimera, and the identification result is A101 (6-7%¥B 101. This phenomenon has been reported in similar study before. This may be related to the cell exchange between the fetus and the mother, organ transplantation, atc. The specific reasons need to be confirmed by further research, 2.4 New variation site in this example, we find a total of 281 mutation sites, 25 mutation sites of which are located in an exon zone or a splicing zone, may greatly influence the gene function. Al present, we have not checked the exons and other mutation sites outside the splicing zone one by one, but only have checked and interpreted the mutation sites in the exon and splicing zone. Among the 25 sites in the exon zone or splicing zone, five sites ars a new variation site, Currently, these five variation sites have not been reported in the database or other literature. They are ¢.773A>C (~5%), ¢.9177>C at

Exon 7, 6.140 _143delTGGA at Exon 3, ©.38dalA at Exon 2, and ©.38delA at Exon 2, and ¢.28+1G>A at the splicing site. These five variation sites are not reported in the 10006 population frequency database, see Table 4.

The ¢.773A>C mutation located in Exon 7 is found in three samples, but the mutation abundance of this mutation site in the three samples is relatively low, only about 5%, the specific reason is not clear yet. $ The ©.917T>C mulation site is associated with the haplotypes B101 mutation site by directly analyzing the mutation based on reads, and we surmise that this site may be a new genotype in the B subtype. The remaining 4 new variation sites are too far apart from the known typing variation sites on the genome, so we cannot directly obtain accurate haplotypes, need further experiments to obtain accurate haplotypes.

Among them, the three samples containing the ¢.773A>C mutation all include the Q02 haplotype, so the mutation site may be related to a new genotype in the O type.

The further experiments include a nanopore third-generation sequencing long- read sequencing experiment, a specific primer amplification sequencing experiment, and a further serological test. We can determine the haplotype of the new variation site and ís antigen expression can be further determined through these experiments.

Table 4 Details of a new variation site

I: FS mi 40 | HO6ISID 002/001 exon? TI3A>C

In summary, the results obtained from ABO whale-gene sequencing typing on 50 samples adopting the sscond-gensration sequencing technology established by the inventor ars consistent with the resulls obtained by traditional serological identification methods, PCR-SSP and Sanger seguencing typing technology.

The invention adopts a sel of seli~designhed primers and high-throughput second-generstion sequencing technology to capture the fulldength ABO gene, based on the PCR amplification-capturing method. The primer design is predicted to cover at least 80.92% of the zones.

In the actual sample detection, whenever the sequencing depth is sufficient (>1000x}, the coverage of >20x is greater than 98%, and the uniformity and specificity are both greater than 80%, an unique typing result is always obtained , which can meet the ABO genotyping requirements.

Compared with the prior

3 ait, the effect of capturing the full-length ABO gene in the invention is better and better, and the experimental cost and steps thereof are simpler and easier than the probe capture method, and it is more suitable for rapid detection to a large number of samples,

it should be understood that after reading the above described content of the invention, a person skilled in the art can make various changes or modifications to the invention, and the forms equivalent to these also fall within the scope defined by the appended claims of the application,

Claims

CONCLUSIESCONCLUSIONS

1. Kit voor ABO-hlosdgroepgenctypering op basis van sequeneren met hoge verwerkingscapaciteit door het vastleggen van meervoudige PCR, die een primerset omvat die bestaat uit primers met de nuclectidesequenties die respectievslijk in SEQ ID NRJ-SEQ ID NR.248 worden getoond.A kit for high-throughput sequencing-based multiple PCR-based ABO cleavage gene typing kit, comprising a primer set consisting of primers having the nucleotide sequences shown in SEQ ID NOJ-SEQ ID NO.248, respectively.

2. Werkwijze voor ABO-bloedgroepgenotypering op basis van segueneren met hoge verwerkingscapaciteit door het vastleggen van mesrvoudige PCR, die de volgende stappen omvat:A method for ABO blood group genotyping based on high-throughput sequencing by mesr-fold PCR capture, comprising the following steps:

S1. het verkrijgen van DNA-monsters van een individu;S1. obtaining DNA samples from an individual;

S2. het uitvoeren van PCR-amplificatie op het DNA-monster door een primerset die beslaat uit primers met de nucleolidsseguenties die respectievelijk in SEQ ID NRT-SEGQ ID NR.248 worden gstoond, waarbij de doelzonesectie wordt vastgelegd om een DNA-bibliotheek te verkrijgen;S2. performing PCR amplification on the DNA sample by a primer set consisting of primers having the nucleolid sequences shown in SEQ ID NRT-SEGQ ID NO.248, respectively, wherein the target zone section is determined to obtain a DNA library;

S3.het verder amplificeran van de DNA-biblotheek door POR. anpiificatieprimers met sequentie-adapters;S3. Further amplifying the DNA library by POR. amplification primers with sequence adapters;

S4. de DNA-bibliotheek het sequeneren met hoge verwerkingscapacitelt op een liumina-platform laten uitvoeren om sequentiedata te verkrijgen;S4. allowing the DNA library to perform high throughput sequencing on a liumina platform to obtain sequence data;

85. het na voorbewerking van de seguentiedata uilvosren van ABO-bloedgroep- genatypering volgens de volgende subslappen:85. After pre-processing the owl fox sequence data, performing ABO blood group genatyping according to the following subfolders:

551. het vergelijken van de sequentie met de menselijke referentiegenoom met behulp van vergelijkingssoftware, S552. het analyseren van het variatielype dat in het monster aanwezig is en het toevoegen van annolatie-informatie aan de variatislocatie met behulp van annotatiesoftware,551. Comparing the sequence to the human reference genome using comparison software, S552. analyzing the variation type present in the sample and adding annolation information to the variation location using annotation software,

S53. het analyseren van het vergelijkingsresultaat en het annotatieresultaat en het verkrijgen van het haplotype van de varialielocatie van het monster,S53. analyzing the comparison result and the annotation result and obtaining the haplotype of the varial location of the sample,

S54. het vergelijken van het haplolyperesultaat van de monstsrvariatislocatie met de ABQ-bloedgroepsysteemdatabase om de ABO- bloedgroeptypering te verkrijgen.S54. comparing the haplolype result of the sample variitis location with the ABQ blood group system database to obtain the ABO blood group typing.

3. Werkwijze volgens conclusie 2 waarbij de werkwijze verder een stap omvalt van het zuiveren van de DNA-bibliotheek met magnetische korrels lussen 82 en 83.The method of claim 2 wherein the method further includes a step of purifying the DNA library with magnetic beads loops 82 and 83.

4. Werkwijze volgens conclusie 2 waarbij de werkwijze verder een stap omvat van het zuiveren van de geamplificeerde DNA-bibliotheek met behulp van tweasiaps magnstische korrelzuivering tussen stappen S3 en 84.The method of claim 2, wherein the method further comprises a step of purifying the amplified DNA library using two-pass magnetic grain purification between steps S3 and 84.

5. Werkwijze volgens conclusie 2 waarbij de werkwijze verder een stap omvat van het uitvoeren van kwaliteitsinspectie met gelelekiroforese op de bibliotheek voor stap 83, en indien de hoofdgeleleldroforeseband ongeveer 400 bp is dan is de kwaliteitsinspactie gekwalficeerd.The method of claim 2 wherein the method further comprises a step of performing gel electrophoresis quality inspection on the library before step 83, and if the main gel electrophoresis band is about 400 bp then the quality inspection is qualified.

©. Werkwijze volgens conclusie 2 waarbij de voorbewerking in stap S5 betrekking heeft op het uitvoeren van kweliteitsinspectie en het fileren van de segusntiedata, het verwijderen van sequentiedata met lage kwaliteit en het verwerken van adaptersequentiedata. ©. The method of claim 2 wherein the pre-processing in step S5 involves performing quality inspection and filtering the sequence data, removing low quality sequence data and processing adapter sequence data.

7 Werkwijze volgens conclusie 2 waarbij de vergslijkingssoftware BWA-software is.The method of claim 2 wherein the comparison software is BWA software.

8. Werkwijze volgens conclusie 2 waarbij de annotatiesoftware annovar-softwara is. The method of claim 2 wherein the annotation software is annovar software.

9, Werkwijze volgens conclusie 2 waarbij de ABO-bloedgroepsysteemdatabase ABO-blosdgrospdata in dbRBC en ISBT-databases omvat, alsmede andere bestaande ABO-bloedgroepdata. The method of claim 2 wherein the ABO blood group system database includes ABO blosdgrosp data in dbRBC and ISBT databases, as well as other existing ABO blood group data.

10, Werkwijze volgens conclusie 2 waarbij indien het In substap S54 verkregen ABO-bloedgroepgsnotyperingresuliaat niet uniek is, de werkwijze verder een stap omvat van het analyseren van de populatiefreguentie van de kandidaattypering van de ABO-bloedgroep om het meest waarschijnlijke typeringsresultaat van de ABO- bloedgroep te verkrijgen.The method of claim 2 wherein if the ABO blood group genotyping result obtained in substep S54 is not unique, the method further comprises a step of analyzing the population frequency of the candidate typing of the ABO blood group to determine the most probable typing result of the ABO blood group to obtain.