N. Yu M. Ohashi S. Alosco M. Salazar K. Cao M. Fernandez Vina E.J. Yunis

Typing of HlA-B*15 alleles using sequencespecif ic primers

Key words: allele: antigen; HLA-Bl5; PCR-SSP; serology: transplantation

Acknowledgments: This work was supported by the American Red Cross New England Region.

Abstract: We have developed a DNA based typing method to detect 38 known B*15 alleles using sequence-specific primers (PCR-SSP). This method involves 38 primers and 39 PCR-SSP reactions with results that can be obtained in 3 hours. The method is easy, fast and suitable for clinical typing for bone marrow and organ transplantation. We have typed 106 HLA-B15 samples using this method. For hornozygous HLA.Bl5 samples, some B*15 allele combinations need to be resolved by additional PCR reactions not included in this article. The method allows the detection of potential new alleles requiring sequencing for confirmation, and it is useful to resolve unusual serological reaction patterns for different HLA-B15 serological speci- ficities. In addition, it could be used to resolve ambiguous PCR-SSOP typing results and for recognition of mismatches in serologically matched unrelated individuals.

Received 27 February. revised. accepted for publication 25 June 1998

Copyrlght 0 Munksgaard 1998 lissue Antigens . ISSN 0001-2815

Tissue Antigens 1998: 5 2 260-269 Printed In Denmark . All rights reserved

The HLA-B15 family comprises 38 structurally closely related al- leles; many of these cannot be distinguished by serologic methods. For example, the HLA specificities B15 and B70 display extensive serologic cross reactivity and the assignment of the associated fine specificities B62, 63, 75, 76, 77, 71 and 72 may be cumbersome to distinguish. Potentially, T lymphocytes may elicit differential recog- nition of some B*15 alleles. Therefore, typing for B*15 alleles may be important in matching for allogenic solid organ and bone mar- row transplantation. Since serology was and still is one of the most widely used methods in HLA typing for cadaveric solid organ trans- plant due to its quick turn around time, the need for an alternative quick and accurate typing method is increasing. In the last decade, the application of molecular typing methods has largely contributed to a more complete and accurate typing of all WHO serological specificities (1-7). The polymerase chain reaction-sequence-specific primer (PCR-SSP) technique is one of the molecular typing tech- niques suitable for solid organ transplantation (3, 4). HLA-B15 is a specificity found with high frequencies in all ethnic groups (8, 9).

Authors' afflllatlonr:

N. Yul. M. Ohashi'. S. Aiosco'. M. Salazar'. K. Cao'. M. Fernandez Vina'. E.J. Yunis', ' IAmencan Red Cross Blood Services, New England Region, MA. USA,

Vmerican Red Cross National Histocompatibility Laboratory, MD. USA,

'Harvard Medical School, Department of Pathology, Dana Farber Cancer Institute. Department of Immunology of Cancer and AIDS. MA, USA

Corrsspondenca to: Neng Yu. MD American Red Cross Blood

Services. New England Region

HLA Laboratory 180 Rustcran Road Suite 150 Dedham MA 02026 USA Fax: + 1 7 8 1 4 6 1 2 2 6 9

260

sequences of sense and antlsense prlmers

Yu et al : Typing HLA-6'15 alleles using PCR-SSP

Primer name

4N

5

6

7

10

11

12

13

14

18

27

28

1506

1507

15E4

1526N

1528

B

C

E

J

K

P

R

S

uo

Un

v W

X

2

AA

cc EE

FF

GG

1519

1533

Sense (S) or antisense (AS)

S

S

S

s S

S

S

s S

s S

S

S

S

s S

S

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

AS

Primer position Exon

117-133 2

117-133 2

119-136 2

181-199 2

170-188 2

195212 2

180-199 2

84-102 2

219-236 2

220-238 2

180-199 2

211-229 2

7-25 3

3-20 3

21-38 4

8-26 3

172-190 2

44-59 3

76-92 3

92-111 3

216233 3

229-246 3

10-29 3

184-201 3

68-85 3

10-28 3

10-28 3

228-245 3

69-86 3

19-36 3

25-42 3

184-201 3

195-212 3

69-87 3

240-257 3

36-51 3

211-229 4

142-159 3

Primer sequence

Table 1

Reference

g C-CgC-gAETCC-gAg-AgA

gC-CgC-gAg-TCC-gAg-gAC

CgC-gAp-TCC-gAg-gAT.~C

ACC-@-AC A-C AC-AgA-TCT-g

@-gTA-l?g-ggA-CCg-@A-C

AAC-ATg-AAg-gCC-TCC-g Cg

gAC-Cgg-AAC-ACAC Ag-ATC-l?

gAC.gAC-ACC-CAg-l?C-gTp-A

gAC-CgA-gAg-AAC-CTg-CgC

ACC-gag-AgA-ACC-Tg C - g T

gAC-Cgg-AAC-AC A-C Ap-ATC-TA

CAC-AgA-CTT-nCC-gAg-AgA-g

ACACCC.TCC-AgA-ppA-TgT T

TCT-CAC-ACC-CTCCAg-AgC

ACACAT-gTg-ACC-C AC-C AC

C AC-CCT-CC A-gAg-gAT-gTA-A

AGT-All-ggg- ACC-W-AgA-T

gAg-gAggCgCCC-gTC-g

CTT-gCC.gTC-gTA-& C-gg

CgT-TC A-psp-CgA-TgT-AAT-CT

gAg-CCA-CTC-CAC-gCA-CAg

CC A-ggT-ATC-Tg C-ggA-gCg

gCC-ATA-CAT-CCT-CTg-gAT@

CTC-TC A-gCT-gCTCCg-CCT

TCg-TAg-gCg-TCC-Tgg-Tgg

CC A-TAC-ATC-gTC-Tg C-C AA-g

CCA-TAC-ATC-CTC-TgC-C AA-g

C Ap-gTA-TCT-gCg-gAg-CCC

gTC-gTA-ggC-gTA-CTg-gTC

CgT-CgC-Ag C-CgT-AC A-TCg

gCC-CC ACgT-CgC-AgCC Ag

CTC-TCA-gCT-gCT-CCg-CCA

CTC-C Ap-gTA-gg C-TCT-CCA

CgT-CgT-Agg-C gT-ACT-ggT-T

CTTCCC-glTCTC-CAg-gTg

gCCCgT-CCg-gCC-CCA-g

TCTCTgCTC-llC-TCC-AgA-AA

gpp-TgA-TCT-gAgCCg-CCT

* * * * * * * * * * * * * * * * * * * * * * *

* c

* * * * * * *I

* * * *

* * * * * * I*

* * * 1

* * * 1

* * * *

* New primers designed for this study

.* Published primer sequences from references (4. 7. 13-15)

Tissue Antigens 1998: 5 2 260-269 261

Yu et al : Typing HL4-B*15 alleles using PCR-SSP

However, the frequencies of private HLA-B15 antigens or alleles are very different in different ethnic groups. We report a PCR-SSP based typing method that can detect 38 B*15 alleles for all ethnic groups in bone marrow and organ transplantation.

Material and methods

DNA samples

In the present study, we typed 106 samples selected from the follow- ing two groups because they carried B15 subtypes as identified by serology or PCR-SOP using the Lifecodes HLA-B QUICK-TYPE KIT V (Cat#165000): 1) UCLA International Cell Exchange cells, 2) patients and unrelated marrow donors.

DNA extractions

DNA was extracted by a cell lysis method (10-12) and resuspended in oligo buffer: 0.05 M KcI,O.O2 M Tns (PH S.O), 2.5 m~ MgCI2, 0.5% Tween 20.

HLA-B'lS alleles Included In the study and their serologlc speclflcltles

HLA alleles

1501

1502

1503

1504

1505

1506

1507

1508

1509

1510

1511

1512

1513

1514

1515

1516

1517

1518

1519

HLA specificity

862

875

872 (70)

862

862

862

862

862

70

871 (70)

815 (75v)

876

877

876

862

863

863

871 (70)

876

HLA alleles

1520

1521

1522

1523

1524

1525

1526N

1527

1528

1529

1530

1531

1532

1533

1534

1535

1536

1537

1538

HLA specificity

862

875

835

BNM5

862

862

B 'Blank"

862

815

815

875

875

862

815

815

815

Table 2

PCR primers

The primers used to amplify B*15 alleles were synthesized from Oligos Etc. Inc. (Wilsonville, Oregon). Primer sequences are listed in Table 1. Alleles considered in this study are previously published (16). Half of the primer sequences were obtained from published articles (4, 7, 13-15). New primers were designed based on the known DNA sequences previously published (16) and listed on ASH web site (http:\\www.swmed.edu\homepages\ASHI). The B*l536 sequence was obtained from the United States National Marrow Donor Pro- gram.

PCR amplification

The reactions were carried out in a final volume of 20 p1 containing genomic DNA (50-200 ng), dNTPs (0.2 m), 20 pmoles of each speci- fic primer, 4 pmoles of internal control primers, 10% dimethylsulfox- ide (DMSO), 0.5 units of Taq polymerase @omega, Piscataway, NJ and Qiagen, Valencia, CA) and 2 p1 of 1 0 ~ PCR buffer (15 m~ am- monium sulfate, 50 mM Tns-HC1 (PH 8.8), 50 p~ EDTA, 0.01% (w/v) gelatin, 10 mM P-mercaptoethanol, 10% (vh) DMSO and 1.5 mM MgClz (1.0 m~ if DNA is resuspended in Oligo buffer)) (17). The inter- nal controls amplified the third intron of the DRBl gene, 5'-primer C5 5'TGC CAA GTG GAG CAC CCA A3' and 3'-primer C3 5'GCA TCT TGC TCT GTG CAG AT3' gave rise to a 796 base pair (bp) fragment (3). PCR reaction was carried out in GeneAmp PCR system 9600 using the following program: hold at 95°C for 3 min, and then for each of the first 5 cycles, 95°C for 30 s, 65°C for 50 s, and 72°C for 50 s; for each of next 5 cycles, 95°C for 30 s, 62°C for 50 sand 72°C for 50 s; and for each of next 10 cycles, 95°C for 30 s, 60°C for 50 s and 72°C for 50 s; and for each of the last 10 cycles, 95°C for 30 s, 55°C for 50 s and 72°C for 50 s; hold at 72°C for 5 min and then hold at 4°C indefinitely.

Gel electrophoresis

The amplified products were separated by electrophoresis in I X TAE/2% agarose (Life Technologies, Grand Island, NY) gel at 6 Vlcm for 20 min, followed by photography of the ethidium bromide- stained PCR products. The 123 bp DNA ladder (Life Technologies, Grand Island, NY) was used as size marker. B*15 alleles were as- signed by the presence or absence of the correct size band on the gel for each specific primer combination.

Results

We developed a quick typing system to type 38 B*15 alleles util- izing sequence-specific primers. The primers were designed to

262 Tissue Antigens 1998: 5 2 260-269

Yu et al : Typing HU-8'15 alleles using PCR-SSP

8'15 allele typlng primer combinations and speclflcltles - # - 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

1 7

18

19

20

2 1

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

Primers

6+C

28+CC

6+P

l O + P

l O + J

4N+J

4N+C

7+J

6+Uo

6+AA

1506+J

1507+CC

12+cc

7+E

7+w

27+C

6+V

18+P

18+C

6+K

11+Un

l l + S

7+c

Size (bp)

489

517

426

375

579

632

491

568

425

598

227

210

548

446

421

428

642

325

388

643

349

406

427

15E4+1519 210

6+6

7 +P

5+cc

1526N+J

1506+CC

1528+J

12+R

6+EE

6+Z

28+1533

13+GG

6+X

14+R

4N+FF

6+FF

456

364

611

226

206

577

537

484

439

464

483

433

498

656

654

Specificity

1501-1502,1504-1508,1511-1516, 1519-1521,1524-1528, 1531-1536, 1538,4601, 5701

1501, 1504,1507-1508,1511-1512, 1514-1515, 1519,1522.1526N-1528, 1530,1532-1535, 1538

1502. 1513,1520-1521,1525, 1536, 1301, (4408)

1502.1513,1521,3501-3504,3506-3509,3511, (3512). 3514-3515, 3518-3521, (4406), 5104, 5301-5302

1502,1508-1511,1513,1515,1518.1521-1523,1529,1531,1537, 35 (minus 3510, 3513, 3515 & 3516), (4406).

5101-5102, (5103), 5104-5108.5301-5302, 5601-5604, 7801-7802, (8201). A'2604

1503,1509-1510,1518,1523,1529,1537,4802, 5601-5604, (8201)

1503,1518,1523, 1529,3907,4802, 5603

1509,1510,1518, 1521.1523, 1537

1504. (1516). 1302, 1303

1505,1520,1531.1301-1303,5701, 5703, (4408, 5702,5704)

1506, 1527, (8201)

1507

1508,1522

1509

1510, 1537

1511, 5603

1512,1519

1513, 5104, 5301-5302, 5801, (4406)

1513.1516.1523-1524, 5301-5302, 5701, 5801-5802

1514,4408

1516

1517

1516,1521,1523, 3907

1519

1520

1521

1522

1526

1527

1528

(1508,1522), 1529, (0708), 3511, 3521, 5101-5104, 5106, 7801-7802

1530, 5702-5703

1532

1533

1534,1301-1303

1535

1536

1537, 1401-1403, 7301

1538 ~~ ~~ ~

Table 3. Amplification for alleles in the parentheses could be weak or negative depending upon the quality and quantity of the DNA used, because one of the two specific primers is not a perfect match for these alleles.

Tissue Antigens 1998: 52: 260-269 263

Yu et al : Typing HlA-B*15 alleles using PCR-SSP

match nucleotide sequences of 38 B*15 alleles. This typing system was tested by typing 106 samples carrying HLA-B15 subtypes. A minimum of 50 cells known to be negative for each specific reaction were used as negative controls for the study. HLA-B15 specificities and alleles studied are listed in Table 2 (18). The primer combi- nations are listed in Table 3. The hit patterns of primer mixes are shown in Fig. 1. A total of 38 primers and 39 primer mixes were used to type for 38 B*15 alleles.

total concordance of the assignments of the B*15 alleles (the highest percent agreement among participating laboratories) as reported by the UCLA International Cell Exchange (19, 20). There were two homozygous and 28 heterozygous samples. Eight samples of B*1503 were present and all were from African American individ- uals.

B*15 allele typing of clinical specimens

B * l 5 allele typing in 30 cells of the UCLA International Cell were typed as B*15 by pcR-SSp. The

Exchange results are shown in Table 5. Eleven samples typed serologically as B70, and ten were assigned correctly. Seven samples were typed as

Thirty cells of the UCLA International Cell Exchange were typed B62 and 14 samples were typed as HLA-B15 by serology. One indi- as B*15 by the PCR-SSP method described in this article. The re- vidual of B*1522 was typed as B53 by serology. In all samples, sults are listed in Table 4. We identified the alleles B*1501, *1502, allele assignment could be reported. The alleles B*1506, *1515, *1503, *1505, *I 51 0, *I 51 I , *1513, *1516, *1517, *1518, *1519, *1522 were found in addition to the alleles detected in the *1523, *1524, *1530, *1535 and *1537 in this panel. There was f i s t panel.

Fig. 1. B'15allele PCR-SSP typing hit pattern. *: Amplification of this allele could be weak or negative depending upon the quality and quan-

tity of the DNA used, because one of the two specific primers is not a perfect match for this allele.

264 Tlssue Antigens 1998: 5 2 260-269

HLA 815 allele level typlng of 30 UCLA lnternatlonal Cell Exchange cells

Yu et al : Typing HLA-B*15 alleles using PCR-SSP

Table 4 ~~

PCR- PCR- UCLA cell exchange results (consensus or majority agreement)

X ID Serology Serology DNA DNA Race SSP SSP -- -- -- 1 TER875 70/72 60

1 TER876 70/72 81/8101/DT

3 TER877 NM5/5/70/15 60

4 TER878 NM5/5/70/15 55

5 TER883 62

6 TER885 75

7 TERB86 70/72

8 TER889 75

9 TER890 15/62

10 TER891 15/62

11 TER892 75/1511

12 TER898 62

13 TER912 75

14 TER913 70/72

15 TER915 70/72

16 TER916 70/72

17 TER917 62

18 TER918 75

19 TER919 75

20 TER920 4 1

21 TER925 70/72

22 TER928 70/72

23 TER930 70/71

24 TER931 62

25 TER932 70/71

26 TER935 70/71

27 TER936 62

28 TER937 63

29 TER938 63

30 TER939 63

53

6 1

7801

77

44

60

54

57

39

18

35

57

52

60

35

75

8101

8101

35

35

46

4 1

27

50

70/71

7

1503

1503

1523

1523

1501

1502

1503

1502

1524

1524

1511

1501

1502

1503

1503

1503

1505

1502

1502

1502

1503

1503

1510

1530

1518

1510

1535

1517

1516

4001

8101

4001

55

5301

4002

7801

1513

4402

4001

5401

5701

39

18

35

57

5201

4001

3505

4101

8101

8101

3501

3512

4601

4102

2706

5001

1537

Black

Black

Caucasian

Caucasian

Black/Oriental

Filipino

Black

Oriental

Caucasian

Caucasian

Japanese

Caucasian

Hispanic/Chinese

Black

Black

Black

Asian Indian

Filipino

Chinese

Caucasian/Oriental

Black

Black

Black

Hispanic

Chinese

Hispanic

Filipino

Caucasian

Black

1502

1503

1523

1523

1501

1502

1503

1502

1524

1524

1511

1501

1502

1503

1503

1503

1505

1502

1502

1502

1503

1503

1510

1530

1518

1510

1535

1517

1516

4001

8101

40

55

5301

40

78

1513

4402

4001

5401

5701

39

18

35

57

5201

4001

3505

4101

8101

8101

35

35

4601

4102

2706

5001

1537

1517 07new Black 1517 07

8*15 allele typing of a panel with unknown serology

We further tested 42 out of 300 miscellaneous samples whose sero- logical data were not available. These 42 samples were previously typed as B*15 by PCR-SOP This panel contained six additional B*15 alleles: B*1504, *1507, *1512, *1513, *1521 and *1527.

We detected a total of 25 (66%) B*15 alleles. Since we do not have positive controls for all 38 B*15 alleles, some primers were tested for non-B15 HLA specificities, for which these primers would also gwe amplifications. For example, primer mix 6+FF, which would amplify B*1538, was not tested because the DNA was not available.

However, the primer 6 was tested with controls for B*1301 and primer FF for B*1401, because these primers would also amplify those alleles. In addition, certain primer mixes would also amplify HLA specificities other than B15 which would need to be resolved. If the second B antigen of a sample is B35, primer mixes 4 and 5 in Table 3, 1O+P and 1O+J, would ampllfy B35 and several B*15 alleles. For example, B*1502 could be amplified by combinations 1, 3, 4 and 5 and B*1525 by 1 and 3. However, B35 could also be amplified by 4 and 5; thus this method would give an ambiguous typing of B*1502, B*35 or B*l525, B*35. Three examples of het- erozygote B*15 allele level typing results are shown in Fig. 2.

Tissue Antigens 1998: 5 2 260-269 265

Yu et at : Typing HL4-B815 alleles using PCR-SSP

8*15 allele typing in homozygotes homozygous cells cannot be resolved because the B*l513 allele alone would give the same amplification pattern as B*1502 and *1513.

Nine of the 106 samples tested in this analysis were B15 homozygotes. Many homozygous B15 samples can be allele typed. However, certain combinations of B15 homozygotes cannot be resolved by primer mix- es listed in Table 3. For example, the B*1503 and B*1537 combi- nation can be typed; however, B*1502, *1513 and B*1503, *1529

This also applies to B*1503 and B*1529 etc. Additional primer com- binations are needed to resolve those ambiguities, and sometimes a combination of PCR-SOP, PCR-SSP and SBT is required to high res- olution type certain B15 homozygotes. One example of homozygous B15 typing results is shown in Fig. 3.

HIA-B 15 allele typlng of cllnlcal speclmens Table 5

# - I

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

3 1

32

33

34

ID Serology

R122587

R119794

R109637

R115625

R108487

R113195

R111266

R111308

R108261

R107524

R106877

R105473

R104975

R104305

R104670

R104669

R104667

R033885

R102222

R096599

R099859

R 0 9 9 8 6 0

R095004

R095005

R097088

R096796

R095142

R153408

R153233

R151356

R151380

R134113

R099197

R063327

15

70

15

7 O?

15

70

15

15

5/63

70

62

15

70

52/15

15

15

15

70

70

70

70

62

15

70

62

62

15

62

62

62

70

53

15

15

Serology

35/53

35/53

7

7

18

14

5 1

4 1

5 1

58

44

40

44

39

38

45

44

39

38

22

38

49

40

38

7

57

44

17

PCR-SSP PCR-SSP

1517 (63)

1503 (72)

1517 (63)

1503 (72)

1501 (62)

1503 (72)

1517 (63)

1501 (62)

1516 (63)

1503 (72)

1501 (62)

1515 (62)

1503 (72)

1516 (63)

1519 (76)

1519 (76)

1519 (76)

1518 (71)

1501 (62)

1503 (72)

1503 (72)

1501 (62)

1503 (72)

1503 (72)

1524 (62)

1524 (62)

1503 (72)

1501 (62)

1501 (62)

1501 (62)

1503 (72)

1522 (35)

1506 (62) 4601

1518 (71)

1510 (71)

4601

1524 (62)

Serologlcal equivalents are listed in parentheses

266 Tissue Antigens 1998: 5 2 260-269

Yu et a1 : Typing HLA-B'15 alleles using PCR-SSP

Positive reactions 5, 6, 8, 15, 38 B*75 allele 8 '1537 Second B allele B*0702

Positive reactions 8 * 1 6 allele B*1522 Second B allele B*5201

2, 5, 13, 27, 31

Fig. 2. B*15 allele typing of three hetero- zygotes using the reactions listed in Table Positive reactions 1, 2, 4, 5, 36

8'1535 3. Electrophoretic migration direction sh(iwn from

123 lip are slioan 111 column hl. .I\ 796 lip intrr11;1l control is st'cii in al l rriictions

bottom to top. The DNA size 111arlier inci-einrnts of 8'15 allele Second B allele B *35

Tlssue Ailfigens 1998: 52: 260-263 267

Yu et al : Typing HLA-B*lS alleles using PCR-SSP

Positive reactions B*75 allel&

1, 6, 7 , 19, 21 8*7503, 7576

Discussion

Our B15 high resolution typing method was designed to provide B*15 allele information in less than 3 h. Therefore, it is suitable for HLA typing for solid organ transplantation. Since turn around time is one of the key factors for typing for transplantation, any unusual B15 serological reaction can be resolved within 3 h using this method. It can identify B*15 allele mismatches in serologically matched indi- viduals, especially in certain ethnic groups (8,9). Ethnic background influences the frequency of certain B*l5 alleles. Therefore, the rela- tive incidence of different alleles in each ethnic group may indicate potential mismatches in serologically matched individuals (e.g. if al- leles B*1516 and B*1517 have a 50%/50% distribution in Africans, then the probabilities of mismatches in B63 matched individuals are very high). For laboratories using PCR-SSOP, this method can also be used to resolve many B*15 allele-related ambiguities. However, this method cannot resolve many ambiguities inB*l5 homozygotes. After we completed this study, additional B*15 alleles were reported, such as B*1539 and B*l540. The increasing number of new alleles would produce further difficulties for B*15 high resolution typing. In ad-

References

Fig. 3. B*15 allele typing of o n e homozy- gote using the react ions listed in Table 3. Electrophoretic migration direction shown from bottom to top. The DNA size marker increments of 123 bp are shown in column M. A 796 bp inter. nal control is seen in all reactions.

dition, this method is not suitable for large volume typing unless pre- aliquot Master Mixes (containing all reagents needed for PCR except for DNA and Taq polymerase) are used.

The DNA quality is critical for successful B15 high resolution typing. In our hands, the cell lysis method for DNA isolation works best. However, if another DNA extraction method is used, the DNA should be resuspended in Oligo solution.

The pH of the amplification condition is also critical for success- ful amplification. pH 8.5 is a preferred amplification condition in our laboratory A pH less than 8.0 could cause weak and false negative amplification of the internal control or the allele-specific amplicon included in a reaction.

Extra bands can be seen with some primer mixes when using DNA of suboptimal quality. Confirmation of the accurate size of the amplified band can help to discriminate true and false positive reac- tions.

In summary, we developed a B15 high resolution typing method with the following features: 1) intermediate to allele level typing, 2) quick turn around time, 3) inexpensive when compared to PCR- SSOP (small sample number) or sequence based typing, and 4) easy to perform and analyze.

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