CN108624671B - Genotype sequences for HLA typing - Google Patents

Abstract

A set of genotype sequences for HLA typing comprising SEQ ID NO: 1, and optionally, further comprises the nucleotide sequence set forth in SEQ ID NO: 2-10. The genotype sequence for HLA typing enriches HLA database, and can effectively improve typing precision, thereby improving the treatment efficiency of related diseases, such as thalassemia treatment, acute leukemia, bone marrow transplantation and the like.

Description

Genotype sequences for HLA typing

Technical Field

The invention relates to the technical field of HLA (human leukocyte antigen) typing, in particular to a group of genotype sequences for HLA typing.

Background

In the 70 to 80 twentieth century, HLA typing techniques were mainly serology and cytology. Since the 90 s, the development of molecular biology, and in particular the introduction of PCR technology, has led to the widespread use of molecular technology in clinical diagnostics, not to mention HLA typing (WoszczekG, et al, "diagnosis of clinical and molecular (PCR-SSP) technologies of HLA-DR typing in clinical laboratory program". Ann transfer. 1997; 2(1): 39-42). In 1991, the 11 th international HLA topic discussion proposed a DNA typing method of HLA, and with the rapid advance of sequencing technology, the typing method based on DNA sequence has replaced the traditional serological and cytological typing methods. The current DNA typing methods are mainly divided into two types: methods based on nucleic acid sequence recognition and methods based on the configuration of sequence molecules. The methods based on nucleic acid sequence identification mainly include: PCR-RFLP, PCR-SSO, PCR-SSP and PCR-SBT (Annia F, et al. "Overview on HLA and DNA typing method". Biotecylolog i A. adaptor 2005; 22: 91-101). Among them, the PCR-SBT sequencing method is the "gold standard" for HLA typing method recommended by the World Health Organization (WHO).

Either of the above typing techniques relies on the HLA database, i.e., the IMGT/HLA database. The discovery of new HLA genotypes is a means for directly enriching HLA databases, and the discovery is helpful for improving the success rate and the accuracy of hematopoietic stem cell transplantation matching.

The IMGT/HLA database has been enriched from 964 genotypes in the first version of 12 months (V1.0) in 1998 to 15819 genotypes in the 72 th version of 10 months (V3.26) in 2016.

The current discovery of new HLA genotypes relies mainly on experimental techniques, i.e., PCR and Sanger sequencing, 3730 peak patterning. In the prior art, two haplotypes need to be detected respectively, and the detection is long in time consumption and low in efficiency.

Disclosure of Invention

The present application provides a set of genotype sequences for HLA typing, including SEQ ID NO: 1.

Further, also includes SEQ ID NO: 2-10.

Further, also includes SEQ ID NO: 2-10.

Further, also includes SEQ ID NO: 2-10.

Further, also includes SEQ ID NO: 2-10.

Further, also includes SEQ ID NO: 2-10.

Further, also includes SEQ ID NO: 2-10.

Further, also includes SEQ ID NO: 2-10.

Further, also includes SEQ ID NO: 2-10, or a pharmaceutically acceptable salt thereof.

Further, also includes SEQ ID NO: 2-10, or a pharmaceutically acceptable salt thereof.

The genotype sequence for HLA typing enriches HLA database, and can effectively improve typing precision, thereby improving the treatment efficiency of related diseases, such as thalassemia treatment, acute leukemia, bone marrow transplantation and the like.

Drawings

FIG. 1 is a flowchart of a method for verifying a new HLA genotype according to an embodiment of the present invention;

FIG. 2 is a flow chart of an embodiment of the present invention for performing HLA typing analysis using the RCHSBT method;

FIG. 3 is a graph of a sequencing peak of sample S01 in an example of the present invention;

FIG. 4 is a graph of a sequencing peak of sample S02 in an example of the present invention;

FIG. 5 is a graph of a sequencing peak of sample S03 in an example of the present invention;

FIG. 6 is a graph of a sequencing peak of sample S04 in an example of the present invention;

FIG. 7 is a graph of a sequencing peak of sample S05 in an example of the present invention;

FIG. 8 is a graph of a sequencing peak of sample S06 in an example of the present invention;

FIG. 9 is a graph of a sequencing peak of sample S07 in an example of the present invention;

FIG. 10 is a graph of a sequencing peak of sample S08 in an example of the present invention;

FIG. 11 is a graph of a sequencing peak of sample S09 in an example of the present invention;

FIG. 12 is a graph of a sequencing peak of sample S10 in an example of the present invention.

Detailed Description

The embodiment of the invention combines an information analysis method, firstly determines one haplotype, and then determines the other haplotype sequence by utilizing an experimental technology. Compared with the prior method, the method can save half of the time and improve the efficiency by 50 percent. Based on the method, a plurality of new gene sequences are discovered, and the contribution degrees to HLA-A, -B, -C, DRB1 and DQB1 in the current database are 0.82%, 0.67%, 0.91%, 1.52% and 3.07% respectively. The present example shows 10 novel genotype sequences.

The method of the embodiment of the invention is shown in the flow chart of fig. 1, and generally comprises the following steps:

placing a DNA library of which the sample source is a bone marrow or umbilical cord blood sample in a sequencer for sequencing;

performing HLA typing analysis on the sequencing data by using an RCHSBT method;

providing a genotype of a known haploid and a most homologous genotype of an unknown haploid based on the analysis results;

extracting DNA of a sample corresponding to an unknown haploid;

performing PCR amplification to obtain a DNA template sequence of an unknown haploid;

performing Sanger sequencing on the amplification products;

and analyzing the sequencing peak graph.

The method provided by the embodiment of the invention can enrich the IMGT/HLA database, improve the success rate and accuracy of hematopoietic stem cell transplantation and matching, and provide more guarantees for the treatment of thalassemia, acute leukemia and other diseases.

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples were carried out under the conventional conditions, unless otherwise specified.

This example selects 10 new genotype sequences from 150 new genotype sequences for display of the new genotype sequence mining process, two for each gene. The details are given in table 1 below:

TABLE 1

Sample numbering	Genes to which the loci belong	Verification scope
			S01	HLA-A	Exons 1-8
S02	HLA-A	Exons 1-8
			S03	HLA-B	Exons 1-7
S04	HLA-B	Exons 1-7
			S05	HLA-C	Exons 1-7
S06	HLA-C	Exons 1-7
			S07	DQB1	Exons 1-6
S08	DQB1	Exons 1-6
			S09	DRB1	Exons 2-6
S10	DRB1	Exons 2-6

Referring to fig. 1, the steps of this embodiment are generally as follows:

1. and (3) placing the DNA library of the sample source bone marrow or umbilical cord blood sample in a sequencer for sequencing. Specifically, Hiseq2500 is adopted for PE sequencing, the prepared Hiseq Rapid SBS Kit v2 reagent is put into a Hiseq2500 sequencer, initialization is completed, then the library is diluted to a certain concentration and is placed at a corresponding position of the sequencer, the number of sequencing cycles is set, and sequencing is carried out.

2. The Sequencing data were analyzed for HLA typing using the RCHSBT Method (see Hongzhi Cao et al. A Short-Read Multiplex Sequencing Method for Reliable, Cost-Effective and High-Throughput Genotyping in Large-Scale students. hum Mutat,2013:00: 1-6). See figure 2 for detailed analysis steps.

3. The genotype of one of the known haplotypes and the most homologous genotype of one of the unknown haplotypes were determined according to the RCHSBT method, and the results are shown in Table 2 below:

TABLE 2

Sample numbering	Genes to which the loci belong	Proximity typing of unknown haplotypes
			S01	HLA-A	A*11:01:01
S02	HLA-A	A*24:02:01
			S03	HLA-B	B*13:02:01
S04	HLA-B	B*15:11:01
			S05	HLA-C	C*04:01:01
S06	HLA-C	C*07:02:01
			S07	DQB1	DQB1*02:02:01
S08	DQB1	DQB1*05:03:01
			S09	DRB1	DRB1*11:01:01
S10	DRB1	DRB1*15:01:01

4. According to the sample information, extracting the DNA of the unknown haploid corresponding sample, and the detailed steps are as follows:

(1) adding 1000 μ L erythrocyte lysate, fully and uniformly oscillating until the mixed solution is bright, and centrifuging at 12000rpm for 1 min; (2) sucking 700. mu.L of the mixed whole blood into a labeled 2mL EP tube; (3) discarding the supernatant, fastening the opening of the EP pipe downwards on absorbent paper, and discharging residual liquid as much as possible; (4) adding 1000. mu.L of erythrocyte lysate, and repeating (2); (5) discarding the supernatant, placing the EP tube with its opening facing downwards on absorbent paper, and discharging the residual liquid as much as possible; (6) adding 200. mu.L ATL, 20. mu.L proteinase K and 200. mu.L AL, shaking for 15s, and centrifuging for a short time; (7) incubating at 56 deg.C for 15min while shaking every 2-3 min; (8) after the liquid is observed to be clear, the incubation is finished, and the centrifugation is carried out for a short time; (9) adding 200 mu L of absolute ethyl alcohol, and slightly inverting the EP tube for 15-20 times; (10) after short-time centrifugation, the mixture is transferred into a corresponding marked QIAampMiniElute Column and is placed for 1-2min at room temperature; (11) centrifuging at 8000rpm for 1min, discarding the collection tube, and transferring the centrifugal column to a new collection tube; (12) adding 500 μ L AW1, centrifuging at 8000rpm for 1min, sucking liquid from the tube opening of the collecting tube, and continuing use; (13) adding 500 μ L AW2, centrifuging at 8000rpm for 1min, discarding the collection tube, and transferring the column to a new collection tube; (14)14000rpm, centrifuging for 3 min; (15) transferring the column to a new 1.5mL EP tube, opening the cover, and air drying for 2-3 min; (16) taking out a piece of ddH2O pre-warmed at 56 ℃, adding 50 mu L of ddH2O to the center of the centrifugal column, and then standing for 1min at room temperature; (17) centrifuging at 8000rpm for 3min, discarding the centrifugal column, and covering with EP tube for storage; (18) measuring related information of the DNA under an ultraviolet spectrometer, and recording the OD value, the purity and the like; (19) the DNA stock is diluted to about 50 ng/. mu.L according to the OD value to be measured (only DNA at a concentration of 50 ng/. mu.L or more may be stored by dilution, and 50 ng/. mu.L or less may be stored as it is).

5. And carrying out PCR amplification on the extracted DNA, carrying out electrophoresis detection on an amplification result and purifying a PCR product. The method comprises the following specific steps:

(1) PCR amplification, detailed steps are as follows:

preparing a PCR reaction system, wherein the contents comprise template numbers, primer names, annealing temperatures and the like; preparing Mix (without primers and templates), fully oscillating and centrifuging the Mix for a short time after the Mix is prepared, and placing the Mix on ice for later use; subpackaging Mix, adding 22 mu L Mix into a reaction plate (tube) added with the primers and the DNA, covering a rubber mat (tube cover), instantly centrifuging to 2000rpm, and waiting for loading; and (3) operating the PCR machine, setting the annealing temperature according to the PCR reaction system, and operating the PCR program.

(2) Electrophoresis detection comprises the following detailed steps:

taking out the PCR plate after the PCR program is run, and performing instantaneous centrifugation to 2000rpm for electrophoresis; loading electrophoresis, namely, taking 3 mu L of PCR product and 2 mu L of 6 multiplied loading buffer solution to be fully and uniformly mixed, and carrying out electrophoresis detection in 1.5% agarose gel; staining with electrophoresis gel, and soaking in ethidium bromide (0.5 μ g/mL) for 15min or more after electrophoresis; and (3) gel imaging, carefully taking out the dyed electrophoresis gel, quickly transferring the gel to an imaging table, adjusting related parameters on imaging software, and taking a picture for storage after observing that the strip is clear.

(3) PCR products were purified in detail as follows:

preparing a purification table, and compiling reaction holes to be purified into the purification table; and (5) membrane filtration purification, namely moistening the membrane, performing plate-rotating suction filtration, and finally performing washing filtration for the second time.

6. The PCR products were Sanger sequenced in detail as follows:

(1) sequencing reaction, the detailed steps are as follows:

compiling a reaction table, wherein the content comprises the information of a sequencing plate number, a product number, a site and a sequencing primer; preparing Mix (without primers and templates), fully oscillating and centrifuging the Mix for a short time after the Mix is prepared, and placing the Mix on ice for later use; split Mix, add 3 μ L Mix (4 μ L volume of Mix with primers added) to 96-well PCR plate (inlet) and cover with a rubber pad, centrifuge instantaneously to 3000 rpm; adding primers, adding 1.1 mu L of corresponding sequencing primers into a PCR plate, covering a rubber mat, and performing instantaneous centrifugation to 3000rpm for later use; adding template (purified product), centrifuging the purified product at 3000rpm for 1min, adding 1 μ L template to the sequencing reaction plate by using a single (eight) channel pipette, covering a rubber pad, and instantaneously centrifuging to 3000 rpm; and (4) operating the PCR machine, and operating the PCR program according to a sequencing reaction system and program.

(2) And (3) purifying after a sequencing reaction, wherein the detailed steps are as follows:

taking the sequencing product off the machine, and centrifuging at 4000rpm for 1 min; chelating metal ions, adding 2 mu L of EDTA-Na2 into the hole, slightly oscillating to make the liquid drop slide down to the bottom of the hole, and fully mixing with the product; first alcohol precipitation, 33. mu.L of 85% ethanol solution was added to the wells and the gel pads were covered. Vortex oscillating for 3min, centrifuging at 4 deg.C and 3000g for 30 min; performing primary reverse centrifugation, preparing absorbent paper with corresponding thickness, orderly filling the absorbent paper into a basket of the centrifuge, taking off a rubber mat, reversely buckling the reaction plate on the absorbent paper, retaining the rubber mat and the reaction plate in a one-to-one correspondence manner for standby, performing instantaneous centrifugation until 760rpm is reached, taking out the reaction plate, and covering the rubber mat; alcohol precipitation is performed again, 50. mu.L of 70% ethanol solution is added to the reaction plate after the first centrifugation, and the original rubber pad is covered. Vortex oscillating for 2min, centrifuging at 4 deg.C and 3000g for 15 min; performing reverse centrifugation again, preparing absorbent paper with corresponding thickness, orderly filling the absorbent paper into a basket of the centrifuge, removing the rubber pad, reversely buckling the reaction plate on the absorbent paper, performing instantaneous centrifugation to 760rpm, and taking out; air-drying in a dark box, and placing the reaction plate in the dark box for air-drying for 15min to completely volatilize ethanol; dissolving the product, adding 10 mu L of HiDi deionized formamide into each hole of the air-dried reaction plate, dissolving and purifying the product, covering a sealing film, oscillating for 10sec, and centrifuging for a short time; and (3) denaturing the product, placing the reaction plate in a PCR instrument, and denaturing at 96 ℃ for 2min to arrange a 3730xl computer.

(3) And (3) processing the 3730xL computer, wherein the detailed steps are as follows:

checking before loading; setting parameters; loading a sample plate and calling a computer program; and (4) recovering the sample plate, automatically conveying the sample plate to a sample stack after the machine-loading procedure is ended, taking out the sample plate, and replacing the rubber gasket with a sealing film for sealing.

The sequencing peak patterns are shown in sequence in FIGS. 3 to 12.

7. And (3) carrying out peak pattern typing, wherein the detailed steps are as follows:

(1) and downloading the sequencing peak image, auditing the peak image according to the quality control standard, and continuously typing the peak image meeting the quality control standard.

(2) And (3) renaming the sequencing peak map by using Smart Rename software according to a sequencing renaming table to match the peak map file with the serial number of the experimental sample.

(3) Peak plot analysis, the specific steps are as follows: using the latest database version peak image analysis software to import the peak image file which has finished name change and screenshot; checking whether the peak image naming information is consistent with the software identification information (including exons, primers and forward/reverse directions), and carrying out forward and reverse sequencing peak image combination comparison on the same sample at the same site; the initial peak view map is combined with software prompt to modify the position of the recognition error; the blue prompt represents that the forward peak image and the reverse peak image are inconsistent, the red prompt represents that the peak images are inconsistent with the database, the inconsistent places are faithfully judged, the corresponding SNP is changed, all the sample site peak images are determined to be corrected, and the closest type is obtained; and comparing the SNP situation in the peak map with the mutation site annotation or the original peak map in the sample information table for rechecking, and if the SNP positions are consistent, judging that the SNP situation is correct.

(4) The genome sequence splicing comprises the following specific steps: if the mutation point is at the edge of the exon, determining the boundary of the exon to ensure that the mutation point is in the exon; selecting a new gene locus genome fasta file from an IMGT/HLA database, searching a closest type of a new gene, copying and pasting the closest type of the new gene into a new text document named as 'ref.fas'; and opening all qualified peak maps of the samples to be tested by using Seqman software, and marking' ref. The failing partial peak plot was masked, the "-" was removed, and the SNPs were altered with lower case letters. And after the change and the check, assembling, storing the consistency sequence and the haplotype file, and storing the genome sequence of the sample to be detected in a 'fas' format, namely 'sample number-site + low type difference _ gen'. And opening all qualified peak maps of the detection sample by using Seqman software, and marking the genome sequence of the sample to be detected as a reference sequence. And (c) shielding the unqualified partial peak image, removing the negative sign, indicating that the genome sequence of the sample to be detected is correctly spliced if no SNP exists, and re-performing the step c) if the SNP still exists.

(5) And CDS conversion, which comprises the following steps: selecting a new gene locus cDNA sequence fasta file from an IMGT/HLA database, and searching a closest type of a new gene; comparing the CDS sequence of the closest type of the new gene in the step with the genome sequence of the sample to be detected, and checking a comparison result; opening a genome sequence file of a sample to be tested by using BioEdit software and editing, wherein the editing content comprises deletion of non-coding sequences and replacement of introns by 100 'N', and storing a CDS sequence of the sample to be tested in a 'fas' format after editing is finished, wherein the CDS sequence is named as 'sample number-site + low type-specific _ nuc'; and repeating the second step, namely comparing the CDS sequence of the sample to be detected with the genome sequence, if the exon where the SNP is located shows that the consistency is 1, indicating that the CDS is converted correctly, and if the exon where the SNP is located is less than 1, converting the CDS again.

(6) According to the above steps, the finally verified new genotype CDS sequence is obtained, which is detailed in table 3 below:

TABLE 3

Remarking: the mutation sites are indicated in bold, italics and underline.

8. Application for the Accession Number.

9. The IMGT/HLA database was submitted.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

SEQUENCE LISTING

<110> Shenzhen Hua Dagen shares GmbH

<120> genotype sequence for HLA typing

<130> 17I24004

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 1098

<212> DNA

<213> genotype sequence S01

<400> 1

atggccgtca tggcgccccg aaccctcctc ctgctactct cgggggccct ggccctgacc 60

cagacctggg cgggctccca ctccatgagg tatttctaca cctccgtgtc ccggcctggc 120

cgcggggagc cccgcttcat cgccgtgggc tacgtggacg acacgcagtt cgtgcggttc 180

gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggg 240

ccggagtatt gggaccagga gacacggaat gtgaaggccc agtcacagac tgaccgagtg 300

gacctgggga ccctgcgcgg ctactacaac cagagcgagg acggttctca caccatccag 360

ataatgtatg gctgcgacgt ggggccggac gggcgcttcc tccgcgggta ccggcaggac 420

gcctacgacg gcaaggatta catcgccctg aacgaggacc tgcgctcttg gaccgcggcg 480

gacatggcag ctcagatcac caagcgcaag tgggaggcgg cccatgcggc ggagcagcag 540

agagcctacc tggagggccg gtgcgtggag tggctccgca gatacctgga aaacgggaag 600

gagacgctgc agcgcacgga cccccccaag acacatatga cccaccaccc catctctgac 660

catgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720

tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca 780

ggggatggaa ccttccagaa gtgggcggct gtggtggtgc cttctggaga ggagcagaga 840

tacacctgcc atgtgcagca tgagggtctg cccaagcccc tcaccctgag atgggagctg 900

tcttcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct ccttggagct 960

gtgatcactg gagctgtggt cgctgccgtg atgtggagga ggaagagctc agatagaaaa 1020

ggagggagtt acactcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc 1080

acagcttgta aagtgtga 1098

<210> 2

<211> 1098

<212> DNA

<213> genotype sequence S02

<400> 2

atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggccct ggccctgacc 60

cagacctggg caggctccca ctccatgagg tatttctcca catccgtgtc ccggcccggc 120

cgcggggagc cccgcttcat cgccgtgggc tacgtggacg acacgcagtt cgtgcggttc 180

gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggg 240

ccggagtatt gggacgagga gacagggaaa gtgaaggccc actcacagac tgaccgagag 300

aacctgcgga tcgcgctccg ctactacaac cagagcgagg ccggttctca caccctccag 360

atgatgtttg gctgcgacgt ggggtcggac gggcgcttcc tccgcgggta ccaccagtac 420

gcctacgacg gcaaggatta catcgccctg aaagaggacc tgcgctcttg gaccgcggcg 480

gacatggcgg ctcagatcac caagcgcaag tgggaggcgg cccatgtggc ggagcagcag 540

agagcctacc tggagggcac gtgcgtggac gggctccgca gatacctgga gaacgggaag 600

gagacgctgc agcgcacgga cccccccaag acacatatga cccaccaccc catctctgac 660

catgaggcca ctctgagatg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720

tggcagcggg atggggagga ccagacccag gacacggagc ttgtggagac caggcctgca 780

ggggatggaa ccttccagaa gtgggcagct gtggtggtac cttctggaga ggagcagaga 840

tacacctgcc atgtgtagca tgagggtctg cccaagcccc tcaccctgag atgggagcca 900

tcttcccagc ccaccgtccc catcgtgggc atcattgctg gcctggttct ccttggagct 960

gtgatcactg gagctgtggt cgctgctgtg atgtggagga ggaacagctc agatagaaaa 1020

ggagggagct actctcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc 1080

acagcttgta aagtgtga 1098

<210> 3

<211> 1089

<212> DNA

<213> genotype sequence S03

<400> 3

atgcgggtca cggcgccccg aaccctcctc ctgctgctct ggggggcagt ggccctgacc 60

gagacctggg ccggctccca ctccatgagg tatttctaca ccgccatgtc ccggcccggc 120

cgcggggagc cccgcttcat caccgtgggc tacgtggacg acacccagtt cgtgaggttc 180

gacagcgacg ccacgagtcc gaggatggcg ccccgggcgc catggataga gcaggagggg 240

ccggagtatt gggaccggga gacacagatc tccaagacca acacacagac ttaccgagag 300

aacctgcgca ccgcgctccg ctactacaac cagagcgagg ccgggtctca cacttggcag 360

acgatgtatg gctgcgacct ggggccggac gggcgcctcc tccgcgggca taaccagtta 420

gcctacgacg gcaaggatta catcgccctg aacgaggacc tgagctcctg gaccgcggcg 480

gacaccgcgg ctcagatcac ccagctcaag tgggaggcgg cccgtgtggc ggagcagctg 540

agagcctacc tggagggcga gtgcgtggag tggctccgca gatacctgga gaacgggaag 600

gagacgctgc agcgcgcgga ccccccaaag acacacgtga cccaccaccc catctctgac 660

catgaggcca tcctgaggtg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720

tggcagcggg atggcgagga ccaaactcag gacactgagc ttgtggagac cagaccagca 780

ggagatagaa ccttccagaa gtgggcagct gtggtggtgc cttctggaga agagcagaga 840

tacacatgcc atgtacagca tgaggggctg ccgaagcccc tcaccctgag atgggagcca 900

tcttcccagt ccaccgtccc catcgtgggc attgttgctg gcctggctgt cctagcagtt 960

gtggtcatcg gagctgtggt cgctgctgtg atgtgtagga ggaagagctc aggtggaaaa 1020

ggagggagct actctcaggc tgcgtgcagc gacagtgccc agggctctga tgtgtctctc 1080

acagcttga 1089

<210> 4

<211> 1089

<212> DNA

<213> genotype sequence S04

<400> 4

atgcgggtca cggcgccccg aaccgtcctc ctgctgctct cgggagccct ggccctgacc 60

gagacctggg ccggctccca ctccatgagg tatttctaca ccgccatgtc ccggcccggc 120

cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacccagtt cgtgaggttc 180

gacagcgacg ccgcgagtcc gaggatggcg ccccgggcgc catggataga gcaggagggg 240

ccggagtatt gggaccggaa cacacagatc tacaagacca acacacagac ttaccgagag 300

agcctgcgga acctgcgcgg ctactacaac cagagcgagg ccgggtctca caccctccag 360

aggatgtacg gctgcgacgt ggggccggac gggcgcctcc tccgcgggca tgaccagtcc 420

gcctacgacg gcaaggatta catcgccctg aacgaggacc tgagctcctg gaccgcggcg 480

gacacggcgg ctcagatcac ccagcgcaag tgggaggcgg cccgtgaggc ggagcagtgg 540

agagcctacc tggagggcct gtgcgtggag tggctccgca gatacctgga gaacgggaag 600

gagacgctgc agcgcgcgga ccccccaaag acacatgtga cccaccaccc catctctgac 660

catgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720

tggcagcggg atggcgagga ccaaactcag gacaccgagc ttgtggagac cagaccagca 780

ggagatagaa ccttccagaa gtgggcagct gtggtggtgc cttctgcaga agagcagaga 840

tacacatgcc atgtacagca tgaggggctg ccgaagcccc tcaccctgag atgggagcca 900

tcttcccagt ccaccatccc catcgtgggc attgttgctg gcctggctgt cctagcagtt 960

gtggtcatcg gagctgtggt cgctactgtg atgtgtagga ggaagagctc aggtggaaaa 1020

ggagggagct actctcaggc tgcgtccagc gacagtgccc agggctctga tgtgtctctc 1080

acagcttga 1089

<210> 5

<211> 1096

<212> DNA

<213> genotype sequence S05

<400> 5

atgcgggtca tggcgccccg aaccctcatc ctgctgctct cgggagccct ggccctgacc 60

gagacctggg ccggctccca ctccatgagg tatttctcca catccgtgtc ctggcccggc 120

cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacgcagtt cgtgcggttc 180

gacagcgacg ccgcgagtcc aagaggggag ccgcgggagc cgtgggtgga gcaggagggg 240

ccggagtatt gggaccggga gacacagaag tacaagcgcc aggcacaggc tgaccgagtg 300

aacctgcgga aactgcgcgg ctactacaac cagagcgagg acgggtctca caccctccag 360

aggatgtttg gctgcgacct ggggccggac gggcgcctcc tccgcgggta taaccagttc 420

gcctacgacg gcaaggatta catcgccctg aacgaggatc tgcgctcctg gaccgccgcg 480

gacacggcgg ctcagatcac ccagcgcaag tgggaggcgg cccgtgaggc ggagcagcgg 540

agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag 600

gagacgctgc agcgcgcgga acacccaaag acacacgtga cccaccatcc cgtctctgac 660

cataaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720

tggcagtggg atggggagga ccaaactcag gacaccgagc ttgtggagac caggccagca 780

ggagatggaa ccttccagaa gtgggcagct gtggtggtgc cttctggaga agagcagaga 840

tacacgtgcc atgttcagca cgaggggctg ccggagcccc tcaccctgag atggaagccg 900

tcttcccagc ccaccatccc catcgtgggc atcgttgctg gcctggctgt cctggctgtc 960

ctagctgtcc taggagctat ggtggctgtt gtgatgtgta ggaggaagag ctcaggtgga 1020

aaaggaggga gctgctctca ggctgcgtcc agcaacagtg cccagggctc tgatgagtct 1080

ctcatcgctt gtaaag 1096

<210> 6

<211> 1084

<212> DNA

<213> genotype sequence S06

<400> 6

atgcgggtca tggcgccccg agccctcctc ctgctgctct cgggaggcct ggccctgacc 60

gagacctggg cctgctccca ctccatgagg tatttcgaca ccgccgtgtc ccggcccggc 120

cgcggagagc cccgcttcat ctcagtgggc tacgtggacg acacgcagtt cgtgcggttc 180

gacagcgacg ccgcgagtcc gagaggggag ccgcgggcgc cgtgggtgga gcaggagggg 240

ccggagtatt gggaccggga gacacagaag tacaagcgcc aggcacaggc tgaccgagtg 300

agcctgcgga acctgcgcgg ctactacaac cagagcgagg acgggtctca caccctccag 360

aggatgtctg gctgcgacct ggggcccgac gggcgcctcc tccgcgggta tgaccagtcc 420

gcctacgacg gcaaggatta catcgccctg aacgaggacc tgcgctcctg gaccgccgcg 480

gacaccgcgg ctcagatcac ccagcgcaag ttggaggcgg cccgtgcggc ggagcagctg 540

agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag 600

gagacgctgc agcgcgcaga acccccaaag acacacgtga cccaccacca tgaggccacc 660

ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 720

ggggaggacc agacccagga caccgagctt gtggagacca ggccagcagg agatggaacc 780

ttccagaagt gggcagctgt ggtggtgcct tctggacaag agcagagata cacgtgccat 840

atgcagcacg aggggctgca agagcccctc accctgagct gggagccatc ttcccagccc 900

accatcccca tcatgggcat cgttgctggc ctggctgtcc tggttgtcct agctgtcctt 960

ggagctgtgg tcaccgctat gatgtgtagg aggaagagct caggtggaaa aggagggagc 1020

tgctctcagg ctgcgtgcag caacagtgcc cagggctctg atgagtctct catcacttgt 1080

aaag 1084

<210> 7

<211> 786

<212> DNA

<213> genotype sequence S07

<400> 7

atgtcttgga aaaaggcttt gcggatcccc ggaggccttc gggcagcaac tgtgaccttg 60

atgctgtcga tgctgagcac cccagtggct gagggcagag actctcccga ggatttcgtg 120

taccagttta agggcatgtg ctacttcacc aacgggacag agcgcgtgcg tcttgtgagc 180

agaagcatct ataaccgaga agagatcgtg cgcttcgaca gcgacgtggg ggagttccgg 240

gcggtgacgc tgctggggct gcctgccgcc gagtactgga acagccagaa ggacatcctg 300

gagaggaaac gggcggcggt ggacagggtg tgcagacaca actaccagtt ggagctccgc 360

acgaccttgc agcggcgagt ggagcccaca gtgaccatct ccccatccag gacagaggcc 420

ctcaaccacc acaacctgct ggtctgctcg gtgacagatt tctatccagc ccagatcaaa 480

gtccggtggt ttcagaatgg ccaggaggag acagctggcg ttgtgtccac cccccttatt 540

aggaatggtg actggacctt ccagatcctg gtgatgctgg aaatgactcc ccagcgtgga 600

gacgtctaca cctgccacgt ggagcacccc agcctccaga gccccatcac cgtggagtgg 660

cgggctcaat ctgaatctgc ccagagcaag atgctgagtg gcattggagg cttcgtgctg 720

gggctgatct tcctcgggct gggccttatc atccatcaca ggagtcagaa agggctcctg 780

cactga 786

<210> 8

<211> 810

<212> DNA

<213> genotype sequence S08

<400> 8

atgtcttgga agaagtcttt gcggatcccc ggagaccttc gggtagcaac tgtcaccttg 60

atgctggcga tcctgagctc ctcactggct gagggcagag actctcccga ggatttcgtg 120

taccagttta agggcctgtg ctacttcacc aacgggacgg agcgcgtgcg gggtgtgacc 180

agacacatct ataaccgaga ggagtacgtg cgcttcgaca gcgacgtggg ggtgtatcgg 240

gcggtgacgc cgcaggggcg gcctgacgcc gagtactgga acagccagaa ggaagtcctg 300

gagggggccc gggcgtcggt ggacagagtg tgcagacaca actacgaggt ggcgtaccgc 360

gggatcctgc agaggagagt ggagcccaca gtgaccatct ccccatccag gacagaggcc 420

ctcaaccacc acaacctgct gatctgctcg gtgacagatt tctatccaag ccagatcaaa 480

gtccggtggt ttcggaatga tcaggaggag acagccggcg ttgtgtccac ccccctcatt 540

aggaacggtg actggacctt ccagatcctg gtgatgctgg aaatgactcc ccagcgtgga 600

gatgtctaca cctgccacat ggagcacccc agcctccaga gccccatcac cgtggagtgg 660

cgggctcagt ctgaatctgc ccagagcaag atgctgagtg gcgttggagg cttcgtgctg 720

gggctgatct tccttgggct tggccttatc atccgtcaaa ggagtcggaa aggacctcaa 780

gggcctccac cagcagggct tctgcactga 810

<210> 9

<211> 701

<212> DNA

<213> genotype sequence S09

<400> 9

cacgtttctt ggagtactct acgtctgagt gtcatttctt caatgggacg gagcgggtgc 60

ggttcctgga cagatacttc tataaccaag aggagtacgt gcgcttcgac agcgacgtgg 120

gggagttccg ggcggtgacg gagctggggc ggcctgatga ggagtactgg aacagccaga 180

aggacttcct ggaagacagg cgggccgcgg tggacaccta ctgcagacac aactacgggg 240

ttggtgagag cttcacagtg cagcggcgag tccatcctaa ggtgactgtg tatccttcaa 300

agacccagcc cctgcagcac cacaacctcc tggtctgttc tgtgagtggt ttctatccag 360

gcagcattga agtcaggttg ttccggaatg gccaggaaga gaagactggg gtggtgtcca 420

caggcctgat ccacaatgga gactggacct tccagaccct ggtgatgctg gaaacagttc 480

ctcggagtgg agaggtttac acctgccaag tggagcaccc aagcgtgaca agccctctca 540

cagtggaatg gagagcacgg tctgaatctg cacagagcaa gatgctgagt ggagtcgggg 600

gctttgtgct gggcctgctc ttccttgggg ccgggctgtt catctacttc aggaatcaga 660

aaggacactc tggacttcag ccaagaggat tcctgagctg a 701

<210> 10

<211> 701

<212> DNA

<213> genotype sequence S10

<400> 10

cacgtttcct gtggcagcct aagagggagt gtcatttctt caatgggacg gagcgggtgc 60

ggttcctgga cagatacttc tataaccagg aggagtccgt gcgcttcgac agcgacgtgg 120

gggagttccg ggcggtgacg gagctggggc ggcatgacgc tgagtactgg aacagccaga 180

aggacatcct ggagcaggcg cgggccgcgg tggacaccta ctgcagacac aactacgggg 240

ttgtggagag cttcacagtg cagcggcgag tccaacctaa ggtgactgta tatccttcaa 300

agacccagcc cctgcagcac cacaacctcc tggtctgctc tgtgagtggt ttctatccag 360

gcagcattga agtcaggtgg ttcctgaacg gccaggaaga gaaggctggg atggtgtcca 420

caggcctgat ccagaatgga gactggacct tccagaccct ggtgatgctg gaaacagttc 480

ctcgaagtgg agaggtttac acctgccaag tggagcaccc aagcgtgaca agccctctca 540

cagtggaatg gagagcacgg tctgaatctg cacagagcaa gatgctgagt ggagtcgggg 600

gctttgtgct gggcctgctc ttccttgggg ccgggctgtt catctacttc aggaatcaga 660

aaggacactc tggacttcag ccaacaggat tcctgagctg a 701

Claims (1)

1. A set of genotype sequences for HLA typing comprising SEQ ID NO: 1-SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

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