WO2012000153A1

WO2012000153A1 - High resolution typing method of hla gene based on illumina ga sequencing technology

Info

Publication number: WO2012000153A1
Application number: PCT/CN2010/001835
Authority: WO
Inventors: 李剑; 田埂; 蒋慧
Original assignee: 深圳华大基因科技有限公司
Priority date: 2010-06-30
Filing date: 2010-11-15
Publication date: 2012-01-05
Also published as: CN101921841A; CN101921841B

Abstract

The invention provides a high resolution typing method of HLA gene based on Illumina GA sequencing technology. Also provided are primer indexes used for the method.

Description

High-resolution typing method of HLA gene based on l l l um i na GA sequencing technology

The invention relates to the field of nucleic acid sequencing technology, in particular to the field of PCR sequencing technology. In addition, the present invention also relates to DN A molecular tagging techniques and DNA incomplete disruption strategies. The method of the invention is particularly applicable to second generation sequencing techniques. The method of the present invention relates to a method of typing DNA sequences, particularly a high resolution typing method for HLA genes. Background technique

Human leukocyte antigen (HLA), one of the most highly recognized gene systems discovered to date, is the main gene system that regulates human-specific immune responses and determines individual differences in disease susceptibility. The rejection of allogeneic organ transplantation is closely related. The study found that the higher the degree of HLA-related gene matching between donor and recipient, the higher the resolution and the longer the graft survival time.

HLA-SBT (Sequence Based Typing) is the main method for high-resolution HLA typing. The method amplifies the gene region of the corresponding HLA by PCR, and sequences the amplified product. The sequencing result is classified by a professional typing software, and finally the HLA genotype information of the sample is obtained. It has the characteristics of being intuitive, highly resolved and capable of detecting new alleles.

The current HLA-SBT method is mainly based on the Sanger sequencing method, and the sequencing method cannot directly obtain the sequence information of the haplotype (parent or parent separate sequence information) in the sample, and only the diploid sequence information can be obtained. This brings uncertainty to the HLA typing results (Ambiguity). In order to obtain a definitive result, GSSP (Group Sepecific Sequencing Primer) or sequencing to solve the above problems is required. Based on the Sanger sequencing method, the sequencing flux is small. Take the saturation flux of 4000 Sequencing reactions of ABI's 3730. Sequencer for one day, and the SBT classification of three sites of HLA-A/B/DRB1 in a single sample. It takes 17 sequencing reactions, and a 3730 sequencer can only produce about 240 samples of data per day.

At present, HLA-SBT is mainly classified by professional typing software. The quality of the imported peak image has a great influence on the peak image recognition ability of the typing software. When the software identifies the error, the typed personnel need to find the error in time. Correct the error. At the same time, in order to avoid human error, the classification work of the same batch of samples is often completed independently by two or more people, and the results can be confirmed only after the verification is correct. If the automation of software typing can be achieved, the incidence of errors will be greatly reduced and labor costs will be reduced.

The HLA-SBT experimental steps based on Sanger sequencing include: PCR amplification, PCR product electrophoresis, PCR product purification, sequencing reaction, sequencing reaction product purification, sequencer sequencing, sequencing result typing, and subsequent addition of GSSP, etc. The process is complicated, and during the experiment, different PCR products can not be mixed together, which greatly increases the experimental workload.

HLA-SBT's complex experimental procedures, low throughput and high cost make it difficult to apply to large-scale HLA high-resolution typing projects.

Summary of the invention

Illumina GA sequencing (Illumina's Genome Analyzer sequencer, referred to as Illumina GA) is a DNA sequence analysis using the principle of sequencing while synthesizing, and can detect haplotypes. The final data produced is a series of base sequences. Directly used for direct comparison with the reference sequence in the HLA database, there is no problem of misclassification of the traditional classification software peak map, which is conducive to the automation of software typing.

Illumina GA has a large sequencing throughput, and now an experimental process can generate 50G (50 billion) bases of data, generating an average of 5 billion bases of data per day. High number According to the flux, the number of sequencing sequences can be determined, so that each sequence can obtain high sequencing depth and ensure the reliability of sequencing results.

At present, there is no research on the application of Illumina GA in the field of HLA typing. The present invention is the first to apply Illumina GA sequencing in the field of HLA typing, combined with DNA molecular tagging technology, DNA incomplete disruption and PCR-FREE library PCR sequencing. Technology that achieves low cost, high throughput, high accuracy, high resolution typing of HLA.

Based on the DNA molecular tagging technology, the separate labeling of multi-sample PCR products is realized, so that the Illumina sequencing library construction experiment can pool multiple samples into one domain for simultaneous processing, which greatly simplifies the experimental operation, and finally, each The test results of the sample can be retrieved through its unique index sequence.

The incomplete DNA disruption technique allows Illumina GA to actually measure the length of the PCR product beyond the sequencing length of the sequencer. In the current maximum Illumina GA sequencing of 200 bp, the actual measurable PCR product is over 200 bp in length.

"adapter" or "library adapter" tag technology refers to the addition of different library linkers to multiple sequencing libraries (different library linkers have different composition sequences, and different sequences are called adapter indices). A library tagging technique is constructed by constructing a tag sequencing library, thereby enabling a plurality of different tag sequencing libraries to be mixed and sequenced, and finally the sequencing results of each tag sequencing library can be distinguished from each other.

The use of PCR sequencing methods based on DNA molecular tagging techniques and DNA incomplete disruption strategies can greatly increase the number of uniquely labeled samples while reducing the number of primer tags (Figure 1).

The PCR-FREE library construction method combining the library linker technology refers to directly connecting the library linker to the ends of the DNA fragment in the sequencing library, and the library linker The import process is called PCR-Free library construction because there is no PCR involved. The access method can be ligated using DNA ligase. There is no PCR participation in the whole library construction process, which avoids the inaccuracy of the final result caused by the introduction of errors in PCR during the construction of the pool product library with high sequence similarity.

The invention adopts a PCR molecular sequencing technology based on DNA molecular tagging technology, DNA incomplete interruption and PCR-FREE construction, and the HLA gene target fragment is grouped by the sample to be analyzed by the two-way primer label. Amplification (the maximum length of the PCR product depends on the maximum DNA length that the sequencer can bind. The current maximum DNA length for Illumina GA is 700 bp, which is the length of the original DNA, does not include the length of the library linker), and the resulting PCR product is equivalent. The PCR-Free tag sequencing library was constructed by mixing and incompletely disrupting the DNA. The different tag sequencing libraries obtained from each sample set were equimolar mixed, and all DNA fragments with a fragment length greater than the maximum sequencing length of the sequencer were selectively recovered and subsequently sequenced using an Illumina GA sequencer. The DNA sequence information of each sample can be obtained by screening the adapter index, the primer label and the sequence information of the PCR primers in the sequencing result, and the obtained DNA sequence is assembled and compared with the corresponding database in the IMGT HLA professional database, and finally The HLA genotype of the sample is available.

In one aspect of the invention, a set of primer indices is provided comprising at least 10 pairs, or at least 20 pairs, or at least 30 pairs, or at least 40 pairs of 95 pairs of primer labels shown in Table 1. Or at least 50 pairs, at least 60 pairs, or at least 70 pairs, or at least 80 pairs, or at least 90 pairs, or 9 pairs (or the set of primer labels are 10 - 95 pairs of 95 pairs of primer labels shown in Table 1) (eg 10 - 95 pairs, 20 - 95 pairs, 30 - 95 pairs, 40 - 95 pairs, 50 - 95 pairs, 60 - 95 pairs, 70 - 95 pairs, 80 - 95 pairs, 90 - 95 pairs, or 95 pairs ))), and And

The set of primer tags preferably comprises at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 in 95 pairs of primer labels shown in Table 1. To PI-40, or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, Or PI-91 to PI-95, or a combination of any two or more of them.

According to another aspect of the present invention, there is further provided the use of the primer tag for a PCR sequencing method, wherein, in particular, each pair of primer tags is combined with a PCR primer pair for amplifying a sequence of interest to be tested into a pair of tag primers The 5th ends of the positive and negative PCR primers have (or are optionally joined by a ligation sequence) a forward primer tag and a reverse primer tag, respectively.

In a specific embodiment of the invention, the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably for amplifying HLA-A/B 2, 3, 4 exon and HLA-DRB1 PCR primers for exon 2, preferably the PCR primers are shown in Table 2.

In another aspect of the invention, there is provided a set of tag primers comprising a set of primer tags described above and a PCR primer pair for amplifying a sequence of interest, wherein each pair of primer tags is combined with a PCR primer pair For the label primer, the 5th ends of the forward and reverse PCR primers have (or are optionally joined by a linker sequence) a forward primer label and a reverse primer label, respectively.

In a specific embodiment of the present invention, the PCR primer in the above-described tag primer is a PCR primer for amplifying a specific gene of HLA, preferably for amplifying HLA-A/B 2, 3, 4 PCR primers for the exon and HLA-DRB1 exon 2, preferably the PCR primers are shown in Table 2.

In another embodiment of the invention, the tag primer is used in a PCR sequencing method. In another aspect of the invention, a method of HLA typing is provided, comprising:

1) providing _n samples, n being an integer greater than or equal to 1, the sample preferably being from a mammal, more preferably a human, in particular a human blood sample;

2) divide the n samples to be analyzed into m groups, m is an integer and n > m > l ;

3) Amplification: For each sample, a pair of label primers are used, and in the presence of a template from the sample, PCR amplification is carried out under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of label primers comprises primers The label's forward label primer and reverse label primer (both may be degenerate primers), wherein the forward label primer and the reverse label primer may contain the same or different primer labels; the primers in the label primer pair used for different samples Labels are different from each other;

4) mixing: mixing PCR amplification products of each sample to obtain a PCR product library;

5) interruption: the resulting PCR product library is incompletely interrupted;

6) Database construction: Combine the library linker technology to construct a PCR-Free sequencing library from the interrupted PCR product library, and recover all the lengths of the sequencer from the maximum read length of the sequencer used to the longest DNA length range used by the sequencer used. DNA bands, different library adapters can be added to the library to distinguish different PCR-Free sequencing libraries;

7) Sequencing: The recovered DNA mixture is sequenced using a second-generation sequencing technique, preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000), to obtain the sequence of the interrupted DNA;

8) Stitching: Based on the different library linker sequences of each library and the unique primer tags of each sample, the obtained sequencing results are in one-to-one correspondence with the samples, and the alignment products (for example, Blast, BWA program) are used to locate the PCR products. On the corresponding DNA reference sequence, the complete target nucleic acid is spliced from the sequence of the broken DNA by sequence overlap and linkage. In a specific embodiment of the present invention, in the method described above, the binding library linker technology, constructing a PCR-free sequencing library from the disrupted PCR product library means using m library linkages to 4 a library of m PCR products obtained by adding a linker, wherein each PCR product library uses a different library linker to construct m linker tag sequencing libraries; m linker tag sequencing libraries are equimolarly mixed to construct a mixture Connector label sequencing library. The method in which the library linker is ligated means that the DNA ligase is directly ligated without a PCR program.

In a specific embodiment of the present invention, in the method described above, each pair of primer tags and PCR primer pairs are combined into a pair of tag primers, and the 5th ends of the forward and reverse PCR primers respectively have (or are optionally connected) Sequence ligation) Forward primer tags and reverse primer tags.

In a specific embodiment of the present invention, in the method described above, the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably for amplifying HLA-A/B 2, 3 , PCR primers for exon 4 and HLA-DRB1 exon 2, preferably the PCR primers are shown in Table 2.

In a specific embodiment of the invention, in the method described above, the primer tag is designed for PCR primers, preferably for PCR primers for amplifying specific genes of HLA, more preferably for PCR primers for amplifying HLA-A\B 2, 3, 4 exon 4 and HLA-DRB1 exon 2, in particular PCR primers as shown in Table 2, including in particular Illustrating at least 10 pairs, or at least 20 pairs, or at least 30 pairs, or at least 40 pairs, or at least 50 pairs, at least 60 pairs, or at least 70 pairs, or at least 80 pairs, or at least 90 pairs, of 95 pairs of primer labels, Or 95 pairs (or the set of primer labels are 10 - 95 pairs of 95 pairs of primer labels shown in Table 1 (eg 10 - 95 pairs, 20 - 95 pairs, 30 - 95 pairs, 40 - 95 pairs, 50 - 95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs), and

In a specific embodiment of the present invention, in the method described above, the DNA disruption comprises a chemical disruption method and a physical disruption method, wherein the chemical method comprises an enzyme digestion method, the physical interruption Methods include ultrasonic interrupting methods or mechanical breaking methods. After the DNA was disrupted, the 450-750 bp length fragment was recovered and recovered. The purification recovery purification recovery method includes, but is not limited to, electrophoresis tapping recovery, and may also be magnetic bead recovery.

In a specific embodiment of the present invention, in the method described above, after the DNA is disrupted, DNA of different sets of samples is connected by different library adaptors during the construction of the PCR-Free tag library. Thus, in the subsequent typing step, the obtained sequencing result corresponds to the sample based on the primer label and the linker label used for each sample.

The alignment sequence of each sample is mapped to the known DNA reference sequence of the PCR product by the alignment program, and the complete PCR product sequence is spliced from the sequence of the broken DNA by sequence overlap and linkage.

In another aspect of the invention, an HLA typing method is provided, comprising: sequencing and splicing a sample (especially a blood sample) from a patient using the sequencing method described above, and splicing the sequence with the HLA database HLA-related sequence data alignment (such as IMGT HLA professional database), the 100% match of the sequence alignment results is the HLA-DRB1 genotype of the corresponding sample.

Advantageous effects of the invention The invention provides a high-resolution typing method of HLA gene based on illumina GA sequencing technology, thereby realizing haplotype sequencing, software typing automation, increasing flux of HLA genotyping and reducing cost.

DRAWINGS

Figure 1: Schematic diagram of the PCR product after labeling the primer and adaptor tags. At the time of the experiment, primer tags were simultaneously introduced at both ends of the PCR product of each sample by PCR; a plurality of PCR products with different primer tags were mixed together to construct a sequencing library. During the sequencing library construction process, when multiple sequencing libraries need to be constructed, each sequencing library can be labeled by adding a library linker with a different linker tag. Once the library is constructed, multiple sequencing libraries with different linker tags can be mixed together and sequenced with Illumina GA (primer tags between sequencing libraries labeled with different linkers can be identical). After the sequencing results are obtained, the DNA sequence information of each sample can be obtained by sorting the linker and primer tag sequence information in the sequencing result.

Figure 2: Electrophoresis results of the corresponding exon PCR products of sample No. 1 HLA-A/B/DRB1. From the electropherogram, the PCR product is a series of single bands with a fragment size of 300bp-500bp, wherein lane M is the molecular weight marker. (DL 2000, Takara), Lanes 1-7 are HLA-A/B/DRB1 exons (A2, A3, A4, B2, B3, B4, DRBl-2) PCR amplification products , Negative control (N) without amplified bands. The results for the other samples are similar.

Figure 3: DNA electrophoresis after HLA-Mix interruption (before and after tapping), the tapping area is 450-750 bp. Lane M is a molecular weight marker (NEB-50bp DNA Ladder), lane 1 is the electrophoresis of HLA-Mix before tapping, and lane 2 is the gel of HLA-Mix after tapping.

Figure 4: Screenshot of the sample consensus sequence constructor, which illustrates the splicing of PCR products based on the overlap between primer tags and DNA fragments. The complete sequence.

detailed description

The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

In an embodiment of the present invention, 950 samples of HLA-A/B 2, 3, and 4 are externally displayed using a PCR-based sequencing method based on primer labeling, DNA incomplete disruption, library tagging, and PCR-FREE library construction. Genotyping of HLA-DRB1 exon 2 (PCR product length between 290 bp and 500 bp) demonstrates that the invention enables low cost, high throughput, high accuracy and high resolution HLA genotyping .

Principle: The samples to be analyzed are divided into 10 groups, and primer labels are introduced into the PCR products of HLA-A/B 2, 3, 4 exons and HLA-DRB1 exon 2 by PCR reaction. A sample information that specifically labels the PCR product. The PCR amplification products of the three sites of HLA-A/B/DRB1 in each group were mixed together to obtain a PCR product library; the obtained PCR product library was incompletely interrupted by ultrasound, and different PCRs were constructed. Free tag sequencing library (where each PCR product library uses a different linker to construct 10 tag sequencing libraries); Mix 10 tag sequencing libraries in equimolar to construct a hybrid tag sequencing library, hybrid tag sequencing library via 2 % low melting agarose electrophoresis, tapping purification to recover all DNA bands between 450-750p length. The recovered DNA was sequenced by Illumina GA PE-100. The sequence information of all the tested samples can be found by the library tag and the primer tag sequence, and the sequence of the entire PCR product is assembled by the overlapping and linkage relationship between the reference sequence information of the known DNA fragment and the sequence of the DNA fragment, and then through the HLA. The alignment of the standard database of the corresponding exons of -A/B/DRB1 can assemble the entire sequence of the original PCR product to achieve genotyping of HLA-A/B/DRB1. Example 1

Sample extraction

Use KingFisher Automatic Extractor (please provide supplier information) (Therm (^ company) from 950 known HLA-SBT typing results of blood samples (Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "Chinese Marrow Bank") DNA extraction. The main steps are as follows: Take out 6 deep-well plates and 1 shallow-well plate of Kingfisher automatic extractor. Add a certain amount of matching reagents according to the instructions and mark them well. Place the plate in the appropriate position, select the program "Bioeasy-200ul Blood DNA_KF.msz", press "star" to execute the program for nucleic acid extraction. After the program is finished, collect the lOOul elution product in the plate Elution. For the extracted DNA, prepare the template for the next PCR. Example 2

PCR amplification

The 950 DNAs obtained in the sample extraction step were sequentially numbered 1-950, and were divided into 10 groups of 95 DNAs each labeled HLA-1, HLA-2, HLA-3, HLA-4, HLA-5, HLA-6, HLA-7, HLA-8, HLA-9, HLA-10. For each set of samples, 95 sets of bidirectional primer labels (Table 1) were used to amplify HLA-A/B 2, 3, PCR primers for exon 4 and HLA-DRB1 exon 2 (Table 2) were used to amplify 95 DNA samples, respectively. The PCR reaction was carried out in a 96-well plate with a total of 7 plates, numbered HLA-XP-A2, HLA-XP-A3, HLA-XP-A4, HLA-XP-B2, HLA-XP-B3, HLA-XP- B4 and HLA-XP-DRB1-2 ("X" indicates sample group number information 1/2/3/4/5/6/7/8/9/10, "A2/3/4, B2/3/4 , DRBl-2" indicates the amplified site), one is not set in each plate A negative control for the template was added, and the primer used for the negative control was a primer labeled with PI-1 (Table 1). At the same time of the experiment, the sample group number information and the primer label information corresponding to each sample are recorded.

Primer tag information

-ει-

SC8l00/0l0ZN3/13d CSIOOO/ZTOZ OAV

PI-95 CGACGTAGAGTC CAGTAGCACTAC Hll 95 95+95*n Table 2, PCR primers for amplification of HLA-A/B/DRB1 corresponding exons before primer labeling

D2-F1, D2-F2, D2-F3, D2-F4, D2-F5, D2-F6, D2-F7 for amplification A forward primer for exon 2 of HLA-DRB1, and D2-R is a reverse primer for amplifying exon 2 of HLA-DRB1.

The PCR procedure for HLA-A/B/DRB1 is as follows:

96 2min

95 " 30s -> 60 30s ^72 20s (32cycles)

15 ∞

The PCR reaction system of HLA-A/B is as follows

PI _nf -D2-F7 ( 2pmol/ ul ) l.Oul

PI„ _r -D2-R ( 2pmoI/ ul ) l.Oul

Promega Taq ( 5U/uI ) 0.2ul

DNA (about 20 ng/ul) 5.0ul

ddH ₂ 0 4.8ul

Total 25.0ul

Wherein, PI _nr A/B/D2-F _{1/2/3/4/5/6/7} represents primer 5, and HLA-A/B/DRB1 with the nth forward primer tag sequence (Table 1) at the end F primer, PI _nr A/B/D2-R _2/3/4 denotes primer 5, and the R primer of HLA-A/B/DRB1 with the nth reverse primer tag sequence at the end (here n < 95 ), others and so on. And each sample corresponds to a specific set of PCR primers (PI _n "A/B/D2-F _{1/2/3/4/5/6/7} , PI _n "A/B/D2-R _{2/3/ 4} ).

The PCR reaction was run on a Bio-Rad PTC-200 PCR machine. After the PCR was completed, 2 ul of the PCR product was detected by 1% agarose gel electrophoresis. Figure 2 shows the electrophoresis results of the corresponding exon PCR products of sample No. 1 HLA-A/B/DRB1. The DNA molecular marker is DL 2000 ( Takara). The gel map has a series of single bands with a fragment size of 300bp-500bp, indicating The HLA-A/B/DRB1 exons (A2, A3, A4, B2, B3, B4, DRB1-2) of sample No. 1 were successfully amplified by PCR, and the negative control (N) had no amplified bands. The results of other samples are similar to this. Example 3

PCR product mixing and purification

For the "X" group ("X" is 1/2/3/4/5/6/7/8/9/10) sample, from the 96-well plate HLA-XP-A2 remaining PCR product (negative control) Except) Take 20 ul each in a 3 ml EP tube, labeled HLA-X-A2-Mix, and perform the same operation on the other 6 96-well plates in the "X" group, labeled HLA-X -A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4-Mix and HLA-X-D2-Mix, shake and mix, from HLA-X-A2-Mix, HLA-X-A3-Mix, HLA-X-A4 200 μl of each of -Mix, HLA-X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4-Mix, and HLA-X-D2-Mix are mixed in a 3 ml EP tube, labeled HLA -X-Mix. The 500 ul DNA mixture was purified by Qiagen DNA Purification kit (see the instructions for specific purification steps). The purified 200 ul DNA was determined by Nanodrop 8000 (Thermo Fisher Scientific).

Example 4

Illumina GA sequencing library construction

l.DNA interruption

A total of 5 ug of DNA from each purified HLA-X-Mix was disrupted on Covaris S2 (Covaris) using Covaris microtubes with AFA fiber caps. The breaking conditions are as follows:

Frequency sweep ( frequency sweepin )

2. Purification after interruption

All interrupted products of HLA-X-Mix were recovered and purified by QIAquick PCR Purification Kit (QIAGEN), dissolved in 37.5ul EB (QIAGEN Elution Buffer );

3. End repair response

DNA end-repairing reaction was performed on the purified HLA-X-Mix after the disruption, and the system was as follows (reagents were purchased from Enzymatics):

DNA 37.5 L

H ₂ 0 37.5 μί Polynucleotide Kinase Buffer ( 10x Polynucleotide Kinase Buffer ( B904 ) ) 10

dNTP mixture (10mM each) ( Solution Set ( lOmM each ) ) 4

T4 DNA Polymerase (T4 DNA Polymerase) 5μL

Kleno Fragment (Klenow Fragment) ΙμΙ

T polynucleotide kinase (T4 Polynucleotide Kinase) 5μL·

Total volume 100 μL·

The reaction conditions are: Constant Temperature Mixer ( Thermomixer, Eppendorf)

20 warm bath for 30 min.

The reaction product was recovered by QIAquick PCR Purification Kit and dissolved in 34 μM EB (QIAGEN Elution Buffer).

4. 3' end plus A reaction

The DNA was recovered in the previous step, and the end was added with A reaction. The system was as follows (reagents were purchased from Enzymatics):

DNA obtained in the previous step 32

10x blue buffer ( 10x blue buffer ) 5

dATP (lmM, GE) 10

Klenow (3'-5' exo-) 3 μΐ^ Total volume 50

The reaction conditions were as follows: a thermomixer (thermomixer, Eppendorf) 37t) warm bath for 30 min.

The reaction product was recovered by MiniElute PCR Purification Kit (QIAGEN) and dissolved in 13 μM of hydrazine solution (QIAGEN Elution Buffer).

5. Linking the Illumina GA PCR-Free library adaptor The term "PCR-Free library adaptor" refers to a designed base that functions to assist in the immobilization of DNA molecules on sequencing chips and to provide universal sequencing primers. The binding site of the PCR-Free library linker can be directly ligated to the DNA fragment in the sequencing library by DNA ligase. The introduction process of the linker is called PCR-Free library linker because there is no PCR involved.

The product after adding A is connected to different Illumina GA PCR-Free index

The reaction conditions are: Constant Temperature Mixer ( Thermomixer, Eppendorf) 20 Warm bath for 15 min.

The correspondence between the sample group and the library connector is as follows

The reaction product was purified by Ampure Beads (Beckman Coulter Genomics) After being dissolved in 50 ul of deionized water, the molar concentration of DNA detected by real-time quantitative PCR (QPCR) was as follows:

6. Tapping recycling

HLA-1-Mix, HLA-2-Mix, HLA-3-Mix, HLA-4-Mix, HLA-5-Mix, HLA-6-Mix, HLA-7-Mix, HLA-8-Mix, HLA -9-Mix and HLA-10-Mix were equimolar mixed (final concentration 72.13 nM/ul), labeled HLA-Mix-10, and 30 L HLA-Mix-10 was recovered with 2% low melting agarose gel. The electrophoresis conditions were 100V, 100min. The DNA marker was a 50 bp DNA marker ₀ tapping gel from NEB to recover a DNA fragment of 450-750 bp in length (Fig. 3). The recovered product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN). After purification, the volume was 32 ul. The DNA concentration was 9.96 nM by real-time PCR (QPCR). Example 5

Illumina GA sequencing

According to the QPCR test results, lOpmol DNA was sequenced using the Illumina GA PE-100 program. The specific procedure is detailed in the Illumina GA operating instructions (Illumina GA Π x ). Example 6

Result analysis

The sequencing result of Illumina GA is a series of DNA sequences, which are constructed by looking up the linker sequence, the positive and negative primer tag sequences, and the primer sequences in the sequencing results. Each primer label corresponds to a database of sequencing results of each of the exon PCR products of the sample HLA-A/B/DRB1. The sequencing results of each exon were mapped to the reference sequence of the corresponding exon by BWA (Burrows-Wheeler Aligner) (reference sequence source: http://www.ebi.ac.uk/imgt/hla/) Build consistency across databases

(consistency) sequence, and then the DNA sequence in the database is screened and sequenced for error correction. Corrected DNA sequence through sequence overlap and linkage

The (pair-End linkage) relationship can be assembled into the corresponding sequence of each exon of HLA-A/B/DRBl. The resulting DNA sequence is utilized in the IMGT HLA Professional Database

HLA-A/B/DRBl sequence alignment of corresponding exons, sequence alignment results

The 100% match is the HLA-A/B/DRB1 genotype of the corresponding sample. A screenshot of the exon 2 consensus sequence builder for the HLA-A site of sample No. 1 illustrated in Figure 4 can be seen. For all 950 samples, the results obtained were completely consistent with the original known classification results. The specific results of samples 1-32 are as follows:

Sample No. Original HLA-A/B/DRBl Type

1 A*02:03 A*ll:01 38:02 "48:01 DRB1' "14:54 DRB1 *15:01

2 A*01:01 A*30:01 ^08:01 ^k 13:02 DRB1' ^03:01 DRB1 *07:01

3 A*01:01 A*02:01 B ^v 45:11 ^fe 47:01 DRB1' ^fe 13:02 DRB1 * 15:01

4 A*24:08 A*26:01 ^k 40:01 B ^{J k} 51:01 DRB1'"04:04 DRB1 *09:01

5 A*01:01 A*24:02 B ^{j fc} 54:01 B ^j 5:02 DRB1'"=04:05 DRB1 *09:01

6 A*01:01 A*03:02 B ^J 15:11 ^k 37:01 DRB1' 0:01 DRB1 *14:54

7 A*ll:01 A*30:01 43:02 B 45:18 DRB1' ^04:04 DRB1 *07:01

8 A*01:01 A*02:01 B ^v ^5:03 B ^vk 81:01 DRB1' 1:01 DRB1 *15:01

9 A*02:06 A*31:01 ^k 27:07 B ^{J k} 40:02 DRB1'"03:01 DRB1 *13:02

10 A*01:01 A*66:01 B ^{J fc} 37:01 ^fc 49:01 ORBV ^10:01 DRB1 ^fc 13:02

11 A*01:01 A*03:01 35:01 52:01 DRB1' ^k 01:01 DRB1 "15:02

12 A*ll:01 A*ll:01 B ^j 15:01 B ^J 45:05 DRB1*04:06 DRB1 45:01

13 A*01:01 A*ll:02 ^k 07:02 B ^J 45:02 DRB1'"09:01DRB1' ^fc 15:01

14 A*01:01 A*02:01 ^k 52:01 B ^{J k} 67:01 ORBV "15:02 DRB1' *16:02

15 A*01:01 A*02:05 ^fc 15:17 B*50:01 ORBV ^fe 07:01 DRBl: ^fc 15:01

16 A*01:01 A*ll:01 ^k 37:01 B ^vk 40:02 ORBV 0:01 DRB1*12:02

17 A*24:07 A*32:01 B ^vk 35:05 ^k 40:01 ORBV ^03:01 DRB1 04:05

18 A*ll:01 A*24:02 B ^vk 13:01 B ^{J k} 35:01 ORBV 46:02 DRB1 ^fc 16:02

19 A*ll:01 A*ll:01 B ^{J k} 40:02 B ^v ^55:12 ORBV ^k 04:05 DRB1'"15:01

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DRB1*1201 in HLA-DRB1 • type is not excluded

The possibility of DRB1*1206/1210/1217, DRB1*1454 does not exclude the possibility of DRB1*1401, since the sequence of the above alleles in exon 2 is identical. The same is true for the identical results of exon 2, 3, and 4 in the HLA-A/B locus.

Using the technical route of the present invention, the samples of 950 known HLA-SBT typing results were genotyped at the HLA-A/B/DRB1 locus, and it was found that the typing results obtained by the technical route of the present invention were obtained. It is exactly the same as the original result.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is to be given by the appended claims references

[1]. http://www.ebi.ac.uk/imgt/hla/stats.html

[2]. Tiercy J M. Molecular basis of HLA polymorphism: implications in clinical transplantation. [J]. Transpl Immunol, 2002, 9: 173-180.

[3]. C. Antoine, S. MUIler, A. Cant, et al. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience. 1968-99. [J]. The Lancet, 2003 ,9357:553-560.

[4]. HA Erlich, G. Opelz, J. Hansen, et al. HLA DNA Typing and Transplantation. [J]. Immunity, 2001, 14: 347-356.

[5]. Lillo R, Balas A, Vicario JL, et al. Two new HLA class allele, DPBl*02014, by sequence-based typing. [J]. Tissue Antigens, 2002, 59: 47-48.

[6]. A. Dormoy, N. Froelich . Leisenbach, et al. Mono-allelic ampli Hcation of exons 2-4 using allele group-specific primers for sequence-based typing (SBT) of the HLA-A, -B and - C genes: Preparation and validation of ready-to-use pre-SBT mini-kits. [J]. Tissue Antigens, 2003, 62: 201-216.

[7]. Elaine R. Mardis. The impact of next-generation sequencing technology on genetics. [J]. Trends in Genetics. 2008, 24: 133-141.

[8]. Christian Hoffmannl, Nana Minkahl, Jeremy Leipzig. DNA barcoding and pyrosequencing to identify rare HIV drug resistance mutations. [J]. Nucleic Acids Research, 2007,1-8.

[9]. Shannon J. Odelberg, Robert B. Weiss, Akira Hata. Template-switching during DNA synthesis by Therm us aquatic ws DNA polymerase I. [J]. Nucleic Acids Research.1995, 23:2049-2057.

[10]. Sayer D, Whidborne R, Brestovac B. HLA-DRB1

DNA sequencing based typing: an approach suitable for hig h throughput typing including unrelated bone marrow regist ry donors. [J]. Tissue Antigens. 2001, 57(l): 46-54.

Claims

Rights request

1. An HLA typing method comprising:

1) providing n samples, n being an integer greater than or equal to 1, the sample preferably being from a mammal, more preferably a human, in particular a human blood sample;

5) interruption: the resulting PCR product library is incompletely interrupted;

6) Building a library: Combining the library linker technology, constructing a PCR-Free sequencing library by breaking the PCR product library, you can add different library connectors to the library.

(Adapter ) to distinguish between different PCR-Free sequencing libraries, recovering all DNA bands between the maximum read length of the sequencer used and the longest DNA length range used by the sequencer used, specifically 450-750 bp length range DNA fragment;

7) Sequencing: The recovered DNA mixture is sequenced using a second generation sequencing technique, preferably a Pair-End technique (e.g., Illumina GA, Illumina Hiseq 2000), to obtain a sequence of the broken DNA;

8) Stitching: Based on the different library linker sequences of each library and the unique primer tags of each sample, the obtained sequencing results are in one-to-one correspondence with the samples, and each sequencing sequence is mapped to the PCR product by an alignment program (for example, Blast, BWA program). Corresponding DNA On the reference sequence, the complete target nucleic acid is spliced from the sequence of the interrupted DNA by sequence overlap and linkage;

9) Classification: The sequencing results are compared with the sequence data of HLA-DRB1 exon 2 in the HLA database (such as IMGT HLA professional database). The 100% matching of the sequence alignment results is the HLA-DRB1 genotype of the corresponding sample. do not.

2. The method of claim 1 wherein each pair of primer tags and PCR primer pairs are combined into a pair of tag primers, the 5th ends of the forward and reverse PCR primers having (or optionally joined by a linker sequence) a forward primer tag and Reverse primer label.

3. The method of claim 1, wherein the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably exon 2, 3, 4 for amplifying HLA-A/B and HLA - PCR primer of DRB1 exon 2, more preferably the PCR primer is shown in Table 2.

4. The method of claim 1, wherein the primer tag is designed for PCR primers for amplifying a specific gene of HLA, preferably for exon 2, 3, 4 for amplifying HLA-A/B And PCR primers for exon 2 of HLA-DRB1, particularly PCR primers as shown in Table 2, in particular, the primer tags include at least 10 pairs of 95 pairs of primer tags shown in Table 1, or at least 20 pairs, or at least 30 pairs, or at least 40 pairs, or at least 50 pairs, at least 60 pairs, or at least 70 pairs, or at least 80 pairs, or at least 90 pairs, or 95 pairs (or the set of primer labels by the table) 1 shows 95 - 95 pairs of 95 pairs of primer labels (eg 10 - 95 pairs, 20 - 95 pairs, 30 - 95 pairs, 40 - 95 pairs, 50 - 95 pairs, 60 - 95 pairs, 70 - 95 pairs, 80 - 95 pairs, 90 - 95 pairs, or 95 pairs), and

The set of primer labels preferably comprises at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 in 95 pairs of primer labels shown in Table 1. To PI-40, or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, Or PI-91 to PI-95, or any two of them Or a combination of multiples.

5. The sequencing method of claim 1, wherein the DNA disruption comprises a chemical disruption method and a physical disruption method, wherein the chemical method comprises an enzymatic cleavage method, the physical disruption method comprising an ultrasonic disruption method or Mechanical breaking method.

The sequencing method according to claim 1, wherein the purification and recovery method comprises, but is not limited to, electrophoretic tapping recovery, and magnetic bead recovery.

7. The sequencing method according to claim 1, wherein the binding library linker technology, the PCR product library constructed by breaking the PCR product library refers to m PCR products obtained by using m library linkers to 2) The library plus the adaptor, wherein each PCR product library uses a different library linker to construct m linker tag sequencing libraries; m linker tag sequencing libraries are equimolarly mixed to construct a hybrid linker tag sequencing library. The method of ligating the library linker means that the DNA ligase is directly ligated without using a PCR program.

8. A set of primer labels comprising at least 10 pairs of 95 pairs of primer labels shown in Table 1, or at least 20 pairs, or at least 30 pairs, or at least 40 pairs, or at least 50 pairs, at least 60 pairs, or at least 70 Yes, or at least 80 pairs, or at least 90 pairs, or 95 pairs (or the set of primer labels are 10 - 95 pairs of 95 pairs of primer labels shown in Table 1 (eg, 10 - 95 pairs, 20 - 95 pairs, 30-95 pairs, 40-95 pairs, 50-95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs), and the set of primer labels are preferred The ground includes at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 to PI-40, or PI in 95 pairs of primer labels shown in Table 1. -41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, or PI-91 to PI- 95, or a combination of any two or more of them.

9. Use of a set of primer tags according to claim 8 for a PCR sequencing method, wherein, in particular, each pair of primer tags and a PCR primer for amplifying a sequence of interest to be tested The pairs are combined into a pair of tag primers, and the 5th ends of the forward and reverse PCR primers have (or are optionally joined by a linker sequence) a forward primer tag and a reverse primer tag, respectively.

10. The use according to claim 9, wherein the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably an exon 2, 3, 4 for amplifying HLA-A/B and HLA-DRB1 PCR primers for exon 2, preferably the PCR primers are shown in Table 2.

11. A set of label primers comprising a set of primer tags of claim 8 in combination with PCR primer pairs for amplifying a sequence of interest, wherein each pair of primer tags and PCR primer pairs are combined into a pair of tag primers, positive and negative PCR The 5' ends of the primers each have (or are optionally joined by a ligation sequence) a primer tag.

12. The label primer of claim 11, wherein the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably exon 2, 3, 4 for amplifying HLA-A/B and PCR primers for exon 2 of HLA-DRB1, preferably the PCR primers are shown in Table 2.

13. Use of the tag primer of claim 11 for a PCR sequencing method.