Preparation method of human HLA typing standard substance and HLA typing standard substance library

Abstract

The invention provides a preparation method of a human HLA (human leukocyte antigen) typing standard substance and an HLA typing standard substance library, and relates to the steps of extracting DNA (deoxyribonucleic acid) of a human blood sample, performing sequencing library construction and hybrid capture on the extracted DNA, then performing sequencing, analyzing a sequencing result according to a human HLA second-generation analysis process DAHLAv0.0.3, and outputting a sample HLA typing result and a sample original sequencing sequence. And comparing whether the original sequencing sequence of the corresponding typing is consistent with the reference sequence of the corresponding typing in the IMGT database, and if the sample contains the typing which is not recorded in the human HLA typing standard substance, putting the sample into the human HLA typing standard substance. Compared with the existing HLA typing standard substance, the typing standard substance library prepared by the invention has the advantages of more complete typing types, higher typing resolution and more accurate typing.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to a preparation method of a human HLA typing reference standard and an HLA typing reference standard library. Background Art

[0002] Human leukocyte antigen (HLA) detection products have been applied clinically. Consistent typing can significantly reduce the incidence of postoperative acute rejection (AR) and prolong the survival period of transplant recipients. However, the development of HLA typing detection products depends on the preparation of typing reference standards, especially for HLA typing detection products based on the amplicon method, such as the ALLTYPE™ FASTPLEX™ NGS 11 LOCI FLEX kit (Thermo Fisher), etc. According to the IMGT database report, the diversity of human HLA genes has exceeded 24,000, and the diversity of HLA typing is still being updated. SNP sites are very likely to fall on the primers, especially at the 3' end of the primers. This indicates that there are situations where homozygous genes cannot be amplified, heterozygous single alleles cannot be amplified, and heterozygous two alleles cannot be amplified when detecting HLA gene typing by the PCR method. Therefore, it is very necessary to fully verify the HLA typing detection using commercial reference standards.

[0003] Currently, domestic commercial reference products for HLA are available from Kebo Company, but the number of loci is too small to meet the requirements for product accuracy verification. The University of California, Los Angeles (UCLA) has relatively complete reference standards based on HLA Class I and Class II. However, it is very difficult to purchase these reference standards. At the same time, since the UCLA reference standards are not commercial, the verification method for the typing results of the reference standards mostly uses PCR, which itself has typing result errors, greatly interfering with the development design and accuracy verification process of HLA detection products. Moreover, due to methodological limitations, most reference products only have 4-digit typing results. There is an urgent need for a set of domestic human HLA typing reference standards with accurate and high-resolution typing results to assist in the development of related typing products. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention adopts a method of combining polymorphic capture probes with next-generation sequencing. By detecting cell lines or blood samples with unknown HLA typing, the HLA typing and the original sequencing sequence of the sample are output. Finally, the reference sequence of this typing is exported from the IMGT database and compared with the original sequencing sequence. If the two are consistent, the typing result is considered correct, and the sample is included in the HLA typing reference standard library. Compared with the existing HLA typing reference standards, the reference standard library prepared by the present invention has the advantages of more complete typing types, higher typing resolution, and more accurate typing.

[0005] The present invention provides a method for preparing a human HLA typing reference standard and an HLA typing reference standard library. The present invention is realized through the following technical solutions: In terms of sample typing detection: A human leukocyte antigen (HLA) kit that combines capture probes with next-generation sequencing is used to detect the HLA typing of a single cell line or blood sample, and the typing result and the original sequencing sequence are output; In terms of rechecking the typing result: The reference sequence of the above typing result is output through the IMGT database, and the original sequencing sequence is compared with the reference sequence of the typing. If they are consistent, it is adopted. If they are inconsistent, the original sequencing sequence is compared with the IMGT database reference sequence again, and finally it can be judged which typing the original sequencing sequence belongs to; In terms of the diversity of reference standards: The HLA typing reference standard library of the present invention is formed by collecting and detecting clinical samples, incorporating more HLA Class I and HLA Class II reference standards with different typings, forming a complete and sound HLA typing reference standard library, and meeting the development needs of subsequent multiplex PCR combined with next-generation and third-generation products.

[0006] The human leukocyte antigen (HLA) kit of the present invention that combines probes with capture method enables the entire detection system to have better compatibility with samples of different typings, with more complete typing types, and can output 6-digit typing results, which has a higher resolution than the 4-digit of conventional reference standards, and the typing results cover 11 genes recommended by the guidelines. At the same time, the typing results included in the IMGT database are compared with the original sequencing sequence to recheck the accuracy of the results, while conventional reference standards usually use PCR or multiplex PCR methods, and there is amplification bias at the source, resulting in little significance in comparing the sequencing data with the reference sequence.

[0007] The present invention provides a method for preparing a human HLA typing reference standard, and the preparation method includes the following steps: (1) Extract the DNA of at least one human blood sample, and quantify the extracted DNA; (2) After constructing a sequencing library and performing hybridization capture on a certain amount of DNA, sequence the obtained sequencing library; (3) Perform human HLA analysis on the sequencing results in step (2), and output the sample HLA typing result and the sample original sequencing sequence; (4) Compare whether the original sequencing sequences corresponding to the respective HLA typings in step (3) are consistent with the reference sequences of the corresponding HLA typings in the IMGT database. If there are HLA typings not included in the human HLA typing reference standards, include the sample in the human HLA typing reference standards.

[0008] Further, number the DNA of the corresponding HLA typings included in the human HLA typing reference standards in step (4), record the high-resolution typing results corresponding to the numbers, and add them to the DNA storage buffer.

[0009] Further, the DNA storage buffer is 10 mM Tris-HCl containing 0.1 mM EDTA.

[0010] Further, the human HLA analysis of the sequencing results in step (2) is performed through the human HLA second-generation analysis process DAHLA_v0.0.3.

[0011] The present invention provides a human HLA typing reference standard library, which is prepared by selecting 156 human blood samples and preparing each blood sample according to a method for preparing a human HLA typing reference standard provided by the present invention. The HLA typing reference standards covered by the human HLA typing reference standard library are shown in Table 4.

[0012] Further, the human HLA typing reference standard library can be used for 6-digit typing of all loci of HLA-A, HLA-B, HLA-C, HLA-DPB1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1 and HLA-DPA1 genes.

[0013] In the present invention, the sequencing of the DNA sequencing library can be carried out using common second-generation sequencers on the market, such as the MGISEQ-T7 sequencer and Illumina Nova series sequencers of MGI, other models of sequencers of MGI and Illumina companies, as well as sequencers of other companies.

[0014] Technical effects of the present invention: Compared with the prior art, the main beneficial effects of the present invention are as follows: (1) In terms of sample genotyping detection: The present invention uses polymorphism capture probes combined with the capture method for detection, which has better compatibility with different genotypes, can cover more types of genotypes. At the same time, it can achieve genotyping of all loci at the 6th allele of 11 genes (HLA-A, HLA-B, HLA-C, HLA-DPB1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, and HLA-DPA1). All competing products are at the 4th allele, and the genotyping resolution is higher, covering all genes recommended by the consensus; (2) In terms of rechecking the genotyping results: Based on the compatibility advantage of sample genotyping detection, there is no preference for the original sequencing data of competing products. It can be compared with the genotyping reference sequences included in the database, which can further confirm the accuracy of the genotyping results and can discover new genotypes not included in the database, and the genotyping is more accurate; (3) In terms of the diversity of reference standards: Based on the collected clinical samples and cell lines, 156 samples are stored at -80°C, and the types of genotypes are more complete. Description of the Drawings

[0015] Figure 1 They are the different base pairs of the genotyping reference sequences of DQA1:05:01 and DQA1:05:05.

[0016] Figure 2 They are the first results of rechecking the different base pairs of the original sequencing sequences.

[0017] Figure 3 They are the second results of rechecking the different base pairs of the original sequencing sequences.

[0018] Figure 4 They are the third results of rechecking the different base pairs of the original sequencing sequences.

[0019] Figure 5 They are the fourth results of rechecking the different base pairs of the original sequencing sequences. Detailed Embodiments

[0020] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the present invention.

[0021] Example 1: Preparation Method of Human HLA Genotyping Reference Standard The DNA in the human blood sample is first quantified by a Qubit fluorometer (Thermo Fisher Scientific, Q33226), then a library is constructed and captured using a human leukocyte antigen (HLA) kit (DiAn, G1102301, G1102302, G1102303), and then sequenced on the BGI sequencer T7. The analysis is carried out according to the human HLA second-generation analysis process DAHLA_v0.0.3 to output the sample typing results, the original sequencing sequence of the sample, and the reference sequence in the IMGT database. Finally, it is compared whether the original sequencing sequence corresponding to the typing is consistent with the reference sequence in the IMGT database. If there is a typing not included in the HLA typing library, the sample is included in the HLA typing reference standard library.

[0022] This method also includes the HLA typing reference standard detection process shown below: 1) Extract the DNA in the blood sample, perform nucleic acid quantification with a Qubit fluorometer (Thermo Fisher Scientific, Q33226), take no less than 300 ng of DNA for mechanical fragmentation, construct a library using a human leukocyte antigen (HLA) kit (DiAn, G1102301, G1102302, G1102303), and after purification, perform hybridization capture using the human leukocyte antigen (HLA) kit. After the library is sequenced on the BGI sequencer T7, the analysis is carried out according to the human HLA second-generation analysis process DAHLA_v0.0.3; 2) Typing result analysis: Retrieve the reference sequence in the IMGT database corresponding to the HLA typing in the IMGT database and compare it with the original sequencing sequence; 3) Reference standard preparation: Number the DNA after typing confirmation, add DNA storage buffer (10 mM Tris-HCl; 0.1 mM EDTA), and store it at -80 °C. Record the high-resolution typing results (A*11:01:01, A*30:01:01; B*46:01:01, B*13:02:01; C*01:02:01, C*06:02:01; DPB1*17:01:01, DPB1*05:01:01; DQB1*03:03:02, DQB1*02:02:01) corresponding to this number.

[0023] It should be noted that the BGI sequencer T7 in step 1) of the HLA typing reference standard detection process can be replaced by a sequencer such as Illumina Nova.

[0024] Example 2: Reliability assessment of the HLA typing reference standard detection process In this experiment, 2 UCLA typing reference standards with known typing results were used to illustrate the reliability of the HLA typing reference standard detection process in Example 1.

[0025] 1) Detection: 300 ng of DNA was taken from each of the two UCLA typing standards with known typing results (C1-107, C2-113), and then the detection process constructed in Example 1 was carried out. That is, after the obtained DNA was mechanically fragmented, library construction and capture were carried out using human leukocyte antigen (HLA) kits (Dian, G1102301, G1102302, G1102303) respectively. The library was sequenced on the BGI sequencer T7, and the off-machine data was analyzed using DAHLA_v0.0.3; 2) Analysis: The typing results were completely consistent with the typing results marked on the UCLA typing standards themselves, as shown in Table 1. In Table 1, each gene involves two alleles. For different UCLA standards, when these two alleles are inconsistent and neither is None, it indicates that the gene is heterozygous. For example, for the C1-107 standard, whether it is the C1-107 standard result (one allele of B is 15:17, the other allele of B is 35:03) or the actual C1-107 detection result (one allele of B is 15:17:01, the other allele of B is 35:03:01), the B gene is heterozygous; when one of the alleles is not None and the other allele is None, it means that these two alleles are consistent, that is, it indicates that the gene is homozygous. For example, for the C1-107 standard, whether it is the C1-107 standard result (one allele of A is 02:01, the other allele of A is None) or the actual C1-107 detection result (one allele of A is 2:01:01, the other allele of A is None), the A gene is homozygous.

[0026] Table 1: HLA Typing Accuracy Results Table

[0027] As can be seen from Table 1, the detection process of the human HLA typing standard in Example 1 is reliable, and its detection results are consistent with the results of the UCLA HLA typing standard, and the HLA typing results are accurate.

[0028] Example 3: Error Typing Correction of Competing Product HLA Typing Standards For the HLA typing standard detection process in Example 1, in order to further illustrate the accuracy of this detection process, this experiment designed 160 HLA typing standards from UCLA, and explored the accuracy of the competing product HLA typing results through the HLA typing standard detection process in Example 1.

[0029] 1) Detection: 300 ng of DNA was taken from 160 UCLA standard products with known genotyping results respectively, and then the HLA genotyping standard product detection process in Example 1 was carried out. That is, after the obtained DNA was mechanically fragmented, a human leukocyte antigen (HLA) kit (Dian, G1102301, G1102302, G1102303) was used to construct a library and perform capture. The library was sequenced on an Illumina Nova, and the raw sequencing data was analyzed using DAHLA_v0.0.3; Table 2: Incorrect Genotyping Results of HLA Genotyping Standard Products of Competing Products

[0030] 2) Analysis: As shown in Table 2, the genotyping results of 4 cases were inconsistent with the UCLA results (C1-213, C2-112, C2-203, C2-205). The genotyping reference sequences marked in the UCLA standard products were compared with the genotyping reference sequences detected according to the detection process in Example 1 and the raw sequencing sequences. Taking C2-203 as an example, the HLA genotyping result marked in the UCLA standard product itself was (DQA1*05:01), and the HLA genotyping detected according to the detection process in Example 1 was (DQA1*05:05:01). By comparing the standard reference sequences of these two HLA genotypings (DQA1*05:01 and DQA1*05:05:01), it was found that the different sites were in Exon 1 and Exon 3. At the same time, the different sites of these two genotypings were located by rechecking the raw sequencing data (the so-called rechecking is to compare the raw sequencing data with the IMGT database reference sequence corresponding to the HLA genotyping standard product genotyping). It was found that the raw sequencing data was consistent with the analysis result of the standard product preparation process of the present invention. Therefore, it was determined that the genotyping of (DQA1*05:05:01) was accurate, as Figures 1-5 shown. Among them, Figure 1 are the different bases of the genotyping reference sequences of DQA1:05:01 and DQA1:05:05, with a total of two different bases. Figure 2 is the first result of rechecking the different bases of the raw sequencing sequence. The first different base of the raw sequencing sequence is completely consistent with the genotyping reference sequence of DQA1:05:05. Figure 3 is the second result of rechecking the different bases of the raw sequencing sequence. The first different base of the raw sequencing sequence is inconsistent with the genotyping reference sequence of DQA1:05:01 of the UCLA standard product. Figure 4 is the third result of rechecking the different bases of the raw sequencing sequence. The second different base of the raw sequencing sequence is completely consistent with the genotyping reference sequence of DQA1:05:05. Figure 5For the fourth review result of the differential bases in the original sequencing sequence, the second differential base in the original sequencing sequence is inconsistent with the UCLA standard DQA1:05:01 typing reference sequence. In Table 2, each gene involves two alleles. For different UCLA standards, when these two alleles are inconsistent and neither is None, it indicates that the gene is heterozygous. For example, for the C2-C203 standard, whether it is the C2-C203 standard result (one allele of DQA1 is 1:03:01 and the other allele of DQA1 is 5:01:01) or the C1-107 actual test result (one allele of DQA1 is 1:03:01 and the other allele of DQA1 is 5:05:01), the DQA1 gene is heterozygous; when one of the alleles is not None and the other allele is None, this means that the two alleles are the same, that is, it indicates that the gene is homozygous. For example, for the C2-C112 standard, whether it is the C2-C112 standard result (one allele of DRB3 is 01:01 and the other allele of DRB3 is None) or the C2-C112 actual test result (one allele of DRB3 is 01:62:01 and the other allele of DRB3 is None), the DRB3 gene is homozygous.

[0031] This example shows that the results detected according to the detection process in Example 1 are correct, correcting the misclassification of the UCLA standard. The HLA typing standard result of the HLA typing standard detection process based on Example 1 is more accurate, and it can verify whether the typing of the HLA typing standard with a known typing result is accurate.

[0032] Example 4: Improvement of HLA typing standard resolution Regarding the HLA typing standard detection process in Example 1, in order to further illustrate the resolution of the present invention, this experiment designed 4 standards (as competing products) from UCLA, and through the detection process in Example 1, explored the resolution performance of the competing product HLA typing.

[0033] 1) Detection: 300 ng of DNA was taken from each of the 4 UCLA standards with known typing results (C2-212, C2-214, C2-216, C2-217), mechanically fragmented, and then library construction and capture were carried out using a human leukocyte antigen (HLA) kit (Dian, G1102301, G1102302, G1102303). The library was sequenced on the BGI sequencer T7, and the downloaded data was analyzed using DAHLA_v0.0.3; 2) Analysis: As shown in Table 3, the genotyping results are consistent with those of UCLA. Compared with the HLA genotyping marked by the UCLA standard product, each gene of the present invention can accurately distinguish the 6-digit level, which is higher than the 4-digit level marked in the UCLA standard product; and the first 4 digits are exactly the same as the results of the competing products, and the results are accurate. In Table 3, each gene involves two alleles. For different UCLA standard products, when these two alleles are inconsistent and neither is None, it indicates that the gene is heterozygous. For example, for the C2-C216 standard product, whether it is the result of the C2-C212 standard product (one allele of DPA1 is 1:03 and the other allele of DPA1 is 2:01) or the actual detection result of C2-C212 (one allele of DPA1 is 1:03:01 and the other allele of DPA1 is 2:01:01), the DPA1 gene is heterozygous; when one of the alleles is not None and the other allele is None, it means that these two alleles are the same, that is, it indicates that the gene is homozygous. For example, for the C2-C216 standard product, whether it is the result of the C2-C216 standard product (one allele of DPA1 is 3:01 and the other allele of DPA1 is None) or the actual detection result of C2-C216 (one allele of DPA1 is 3:01:01 and the other allele of DPA1 is None), the DPA1 gene is homozygous.

[0034] Table 3: Improvement of HLA Genotyping Standard Product Genotyping Resolution

[0035] This example shows that the present invention can provide a preparation process and standard product DNA with higher resolution than the existing UCLA standard product, and can directly prepare HLA genotyping standard products with 6-digit genotyping, which is of great help for verifying the performance of HLA genotyping products based on the PCR methodology.

[0036] Example 5: Range of HLA Genotyping Standard Products In order to further illustrate the scope of the products of the present invention, this experiment designed to select 156 samples, and through the preparation process in Example 1, a group of HLA high-resolution standard products was initially formed. The list of HLA genotyping standard products included is shown in Table 4.

[0037] 1) Detection: 300 ng of DNA was taken from 156 UCLA standard products with known genotyping results respectively. After mechanical fragmentation, library construction and capture were carried out respectively using a human leukocyte antigen (HLA) kit (Di'an, G1102301, G1102302, G1102303). The library was sequenced on an illumina Nova, and the off-machine data was analyzed using DAHLA_v0.0.3; 2) Analysis: The typing results correspond one-to-one with the sample numbers, starting from HLA-HQ1, covering all 6-digit typing information of 11 genes including HLA-A / B / C / DPB1 / DQB1 / DRB1 / 3 / 4 / 5 / DQA1 / DPA1; 3) Preparation of the standard sample group: Add TE to the corresponding samples and store them at -80°C.

[0038] This example shows that based on the existing samples, the present invention has prepared a group of high-resolution HLA typing standard samples. The HLA typing standard sample library of the present invention contains a total of 156 samples, covering all typing information of 11 genes, and the resolution of the typing results reaches 6 digits. Subsequently, different typed samples can be continuously incorporated into the HLA typing standard sample library by using this method, as shown in Table 4. In Table 4, Library represents the standard sample library, Gene is the gene, Allele1 is allele 1, and Allele2 is allele 2; for each gene, when Allele1 and Allele2 are inconsistent and neither is None, it indicates that the gene is heterozygous; when Allele1 is not None and Allele2 is None, it means that Allele2 is the same as Allele1, that is, it indicates that the gene is homozygous; when both Allele1 and Allele1 are None, it indicates that the gene does not exist.

[0039] Table 4: List of HLA Typing Standard Sample Library

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

Claims

1. A method for preparing a human HLA typing reference standard, characterized in that, The preparation method shown includes the following steps: (1) Extract the DNA of at least one human blood sample and quantify the extracted DNA; (2) After constructing a sequencing library and performing hybridization capture on a certain amount of DNA, sequence the obtained sequencing library; (3) Perform human HLA analysis on the sequencing results in step (2) and output the sample HLA typing results and the original sequencing sequences of the samples; (4) Compare whether the original sequencing sequences corresponding to the typing in step (3) are consistent with the reference sequences of the corresponding typing in the IMGT database. If there is a typing not included in the human HLA typing reference standard, include this sample in the human HLA typing reference standard.

2. The preparation method according to claim 1, characterized in that, Number the DNA of the corresponding typing included in the human HLA typing reference standard in step (4), record the high-resolution typing results corresponding to this number, and add it to the DNA storage buffer.

3. The preparation method as shown in claim 2, characterized in that, The DNA storage buffer is 10 mM Tris-HCl containing 0.1 mM EDTA.

4. The preparation method as shown in claim 1, characterized in that, The human HLA analysis of the sequencing results in step (2) is performed through the human HLA second-generation analysis process DAHLA_v0.0.

3.

5. The preparation method as claimed in claim 1, characterized in that, The sequencing in step (2) is performed by a next-generation sequencer.

6. A human HLA typing reference standard library, characterized in that, The human HLA typing reference standard library is prepared from 156 human blood samples according to the preparation method in claim 1 for each blood sample.

7. The human HLA typing reference material library as described in claim 6, characterized in that, The human HLA typing reference standard library can be used for 6-digit typing of all loci of HLA-A, HLA-B, HLA-C, HLA-DPB1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, and HLA-DPA1 genes.

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