WO2016129759A1 - Next generation sequencing technique-based high-efficiency, high-resolution histocompatibility typing analysis method and kit - Google Patents

Abstract

The present invention relates to a method and a kit which can simultaneously test HLA-A, B, and DRB1 allele typing through high-resolution analysis using a next generation sequencing (NGS) technique.

Description

High efficiency, high resolution histocompatibility type analysis method and kit based on next generation sequencing technology

The present invention relates to a method and kit that can simultaneously test HLA-A, B, DRB1 allele types by high resolution analysis using Next Generation Sequencing (NGS).

Human Leukocyte Antigen (HLA) is an immune response regulatory gene that produces major factors that cause rejection in transplantation of hematopoietic stem cells (bone marrow) and organs, also known as histocompatibility antigens. When foreign substances such as bone marrow cells or viruses of others enter the body, the human body takes an action to remove them in order to protect itself, and plays an important role in determining whether the substance is the same as itself. It is HLA.

To increase the success rate of bone marrow or organ transplantation, the HLA type of the patient and the donor must be matched.If the HLA is inconsistent, the transplanted hematopoietic stem cells are regarded as a foreign object from the patient and attacked or rejected. Graft-versus-host disease, in which T-lymphocytes treat the patient as a foreign body and attack the patient, may cause symptoms such as skin disease, diarrhea, and liver dysfunction, and in severe cases, death. In both cases, the HLA test is performed before the transplantation of organs and hematopoietic stem cells.

Current HLA test methods are classified into low resolution, middle resolution, and high resolution according to the resolution of the test.

Low-resolution serological methods require highly skilled skills and experience, and inconsistencies in comparison with DNA tests have resulted in DNA tests that are more easily performed in smaller laboratories. It is rapidly replacing.

Sequence specific oligonucleotide probe (SSOP) method used for medium resolution analysis is a method of determining the result based on the hybridization reaction with the probe fixed to the strip after PCR amplification. The SSOP method has the advantage of being somewhat readable due to the small number of ambiguities due to cross reactions and weak reactivity, which are commonly found in serological tests, and the ability to process a large amount of samples at the same time. Because it takes a while, it takes a little time and sometimes it is ambiguous in determining the positive band.

The PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primer) technique, which is used for medium resolution analysis, selectively amplifies genes by using sequence differences of PCR primers, and controls the number of sets of PCR primers to determine the desired level of type. It is a technique to judge. The PCR-SSP method has the advantage that the result can be immediately confirmed through electrophoresis after PCR because only a specific allele group is amplified and determined in the PCR process, but it is difficult to simultaneously test a plurality of samples.

SBT (sequence-based typing), which is used as a high resolution method, is the best method for new type analysis and contributes to the rapid increase in the number of HLA types. However, there is a problem of phase ambiguity that cannot distinguish between cis-type and trans-types of allelic variants, and has a disadvantage of time, labor, and cost.

As reports of poor results based on low resolution typing analyzes accumulate, there is a consensus on the necessity of high resolution analysis.In the case of non-related hematopoietic stem cell transplantation, even if the HLA type is matched at the serological level, if the discrepancy at the high resolution level is rejected, There have been reports of higher rates of acute graft-versus-host disease and mortality (N Engl J Med 1998; 92: 3515-20).

Therefore, the importance of the use of the HLA high resolution analysis is increasing, and the use of the SBT (sequence-based typing) analysis, which is currently the international standard HLA high resolution analysis technology, is required. However, there are disadvantages of high cost, low throughput, inherent limitations of phase ambiguity, and so on, and there are limitations in the application for the hematopoietic stem cell applicant registration database, which requires detection of large HLA assays.

Since 2009, active studies on HLA type analysis using NGS instruments have been conducted, and the results have been significantly reduced in ambiguity compared to the results using conventional high resolution method SBT (sequence-based typing) (Proc Natl Acad Sci US). A. 2012 May 29; 109 (22): 8676-81). However, due to the relatively high sequencing cost using NGS instruments, NGS-based methods cannot be considered to be significantly superior to conventional SBT based HLA analysis methods in terms of throughput and cost. The disadvantage is that it depends on the NGS device. There is still a need to develop an HLA high resolution test method which fundamentally solves the problem of phase ambiguity, which is an inherent limitation of the HLA high resolution method, and is easy to process large samples at low cost.

Therefore, the present inventors conducted a study on a method for efficiently analyzing a large amount of samples using the NLA-based HLA high resolution analysis method, and the cost through the production of a library for NGS by a combination of multiple PCR and Fusion PCR The present invention has been completed by discovering that time can be shortened and dependence on specific NGS devices can be eliminated.

       It is an object of the present invention to provide a method that can simultaneously examine HLA-A, B, and DRB1 allele types by high resolution analysis using next generation sequencing technology (NGS).

It is also an object of the present invention to provide a kit that can be used to simultaneously test HLA-A, B, and DRB1 allele typing with high resolution analysis using NGS.

The present invention also aims to provide a universal primer that can be usefully used for library construction for NGS.

In order to achieve the above object, an aspect of the present invention is a method for performing high resolution histocompatibility (HLA) type analysis using next-generation sequencing (NGS),

Amplifying the exon regions of HLA-A, B, and DRB1 in the sample to obtain a target PCR product,

The obtained target PCR product was used as a template, and a Fusion PCR was performed using barcoded primers including a universal primer sequence for sequence analysis, a barcode sequence for sample identification, and an adapter sequence, thereby performing an adapter for NGS and a barcode for sample identification. To obtain the barcoded PCR product bound thereto,

It provides a method comprising the step of performing the NGS on the barcoded PCR product.

As used herein, the term "Next Generation Sequencing (NGS)" is a technique for fragmentation and rapid and accurate reading of a sequence. Unlike the Sanger method, the term "Next Generation Sequencing (NGS)" is required for genome translation through the production of massively parallel data. It significantly reduces cost and time, and means the technology that is most widely used for genome decoding. In NGS, there are some differences depending on the model, but basically amplify the DNA sequence, and then photograph the fluorescence label with a camera to read the base. Depending on the PCR amplification method, Roche (454) and Life Technologies models can be divided into emulsion PCR (emPCR) method, and those of Illuminaa using solid-phase amplification (Michael, L .. Metzker. (2010) Sequencing technologies-the next generation.Nature Reviews Genetics. 11, 31-46).

The term "high resolution HLA typing analysis" as used herein refers to a method for determining HLA typing by sequence based typing (SBT), and more specifically, HLA typing analysis based on NGS. More specifically, HLA typing can be determined up to subtypes of subclasses of the HLA gene as compared to low resolution or medium resolution analysis by SSOP or PCR-SSP. For example, high resolution HLA type means HLA-A * 02: 06: 01, HLA-A * 26: 02: 01, etc., and medium resolution HLA type means HLA-A * 02: 06, HLA-A * 26 : 02, etc., and low resolution HLA type means HLA-A * 01, HLA-A * 02, and the like.

High resolution HLA typing is required to prevent hematopoietic stem cell transplant rejection and graft-versus-host disease.

As used herein, the term "sequencing universal sequence" or "sequencing universal primer sequence" refers to a primer sequence that is universally used for sequencing regardless of the type of NGS instrument. Examples include, but are not limited to, SP1 and SP2.

As used herein, the term “sample identification barcode” refers to a sequence that connects or binds to a sample to identify each sample when analyzing multiple samples at the same time, and includes a length of an appropriate number of nucleotides as necessary. Can be. The binding of barcode sequences to samples allows for identification between samples, allowing pooling of samples for simultaneous analysis, saving time, money, and labor.

As used herein, the term "adapter" for NGS includes sequences for fixing or binding the DNA of interest to sequencing chips or microparticles for analysis in NGS instruments and universal sequences for sequencing.

As used herein, the term "barcoding primer" means a primer comprising a barcode sequence for sample identification. Barcoding primers may include, in addition to barcode sequences for sample identification, universal primer sequences for sequencing and adapter sequences suitable for the NGS instrument to be used.

As used herein, the term “adapter primer” refers to a primer used to bind an adapter suitable for an NGS instrument to an NGS assay DNA, and consists of an adapter sequence and a universal primer sequence.

As used herein, the term “target PCR” refers to a PCR that amplifies a target region using a primer pair specific for a target region on DNA, and includes a universal primer sequence to which an adapter for NGS can be linked. do.

As used herein, the term “Fusion PCR” or “fusion PCR” refers to a PCR performed to bind or connect an adapter and barcode sequence for NGS to a PCR product obtained by amplifying a target region.

1 is amplified by PCR together with a schematic diagram of the genes of HLA-A and HLA-B belonging to HLA class I and HLA-DRB1 belonging to class II in high resolution HLA typing analysis according to an embodiment of the present invention The target region is shown.

The HLA gene is known as one of the most mutated genes, and HLA typing analysis is an essential test for organ and hematopoietic stem cell transplantation because of its important function of distinguishing self from nonmagnetic. In particular, high-resolution HLA-type analysis is required for non-related hematopoietic stem cells or organ transplants. The number of types known to date is 2,946 for HLA-A, 3,693 for HLA-B, and 1,582 for DRB1 (IMGT / HLA Database, Release 3.18.0) as of October 2014. Therefore, for HLA typing, it is important to design primers that can amplify all types with different sequences and do not cause non-specific amplification. In addition, the annealing temperature may be considered when designing a primer to perform multiple PCR.

In one embodiment of the present invention, amplifying the exon regions of the HLA-A, HLA-B and HLA-DRB1 to obtain a PCR product may be performed by multiplex PCR.

In one embodiment of the present invention, amplifying the exon regions of the HLA-A, HLA-B and HLA-DRB1 to obtain a PCR product is a primer for amplifying the HLA-A of SEQ ID NO: 1 to 6, SEQ ID NO: 7 to It can be carried out using a combination of primers selected from the group consisting of primers for amplifying HLA-B of 12 and primers for amplifying HLA-DR of SEQ ID NOs: 13 and 14.

The primers of SEQ ID NOs: 1 to 14 select a highly conserved base sequence site to amplify all alleles present in the target regions of HLA-A, HLA-B, and HLA-DR, and two at one location It is designed to have a degenerate site having the above base. For example, SEQ ID NO: 13 has six degenerate sites.

In one embodiment of the present invention, the multiplex PCR may amplify exons 2, 3, 4 of HLA-A, exons 2, 3, 4 of HLA-B, and exon 2 of HLA-DRB1.

Each target region, HLA-A exon 2, exon 3, exon 4, HLA-B exon 2, exon 3, exon 4, and exon 2 of HLA-DRB1 can be amplified by separate PCR, but save time and labor These can be amplified by multiplex PCR.

In one embodiment of the present invention, the multiplex PCR amplifies HLA-A-exon 4, HLA-B-exon 2, HLA-B-exon 4 in one PCR reaction, HLA-A-exon 2, HLA- A-Exon 3, HLA-B Exon 3, and HLA-DRB1-Exon 2 can be amplified in one PCR reaction and performed in two multiple PCR reactions.

The ratio of primers to each target can be adjusted so that multiple PCR reactions do not bias against specific targets.

In one embodiment of the present invention, the multiple PCR of the HLA-A-exon 4, HLA-B-exon 2, HLA-B-exon 4 is HLA-A-exon 4, HLA-B-exon 2, HLA-B -The primer of exon 4 can be performed on the conditions which mixed 3: 3: 1.

In one embodiment of the present invention, the multiple PCR of the HLA-A-exon 2, HLA-A-exon 3, HLA-B-exon 3, HLA-DRB1-exon 2 is HLA-A-exon 2, HLA-A Exon 3, HLA-B-exon 3, HLA-DRB1-exon 2 primers can be carried out under mixed conditions of 1: 1: 1: 1.5.

If the amplification products are generated in the same ratio in the target PCR, dilution to equalize the amount of each product in the quantification step before NGS after Fusion PCR can be simplified or eliminated.

In one embodiment of the present invention, the primers for target PCR may include a universal sequence in common so that the adapter including the sample identification barcode sequence can be linked.

The sample identification barcode sequence and universal sequence can be designed to suit NGS instrument characteristics.

In one embodiment of the present invention, the sample identification barcode sequence may be one of SEQ ID NO: 15 to 110.

In one embodiment of the invention, the universal sequence may be SEQ ID NO: 111 or 112.

In one embodiment of the present invention, using the PCR product obtained after the target PCR as a template, performing PCR using a barcode primer comprising a universal sequence for sequencing, a barcode sequence for sample identification, and an adapter sequence for NGS Obtaining the barcoded PCR product in which the adapter and the barcode for identifying the sample are combined may be performed using a barcode primer selected from the group consisting of barcode primers of SEQ ID NOs: 115 to 210 and an adapter primer of SEQ ID NO: 211. .

For sample analysis by NGS, a sample is prepared by combining an adapter for NGS and a barcode for sample identification for analysis of multiple samples.

The barcode sequence acts as a label to identify each sample, allowing multiple barcoded samples to be integrated and analyzed simultaneously.

Unlike conventional bar coding methods for NGS using commercial kits ligating adapters containing barcode sequences to samples, the method of the present invention is a target PCR product in which a target sequence and a universal sequence to which an adapter sequence for NGS is linked are linked. After obtaining, the product was subjected to Fusion PCR with a barcode primer comprising an adapter sequence and a barcode sequence, thereby binding the adapter for NGS and the barcode for sample identification to the target PCR product. Although a unique adapter is currently required for each commercially available NGS device, the present invention can obtain a product for sequencing suitable for an NGS device to be used by linking the adapter sequence required for the NGS device to a target PCR product.

 Thus, the number of samples that can be analyzed at the same time can be increased by increasing the type of barcode sequence as needed, without depending on a particular NGS instrument or kit therefor.

In addition, the barcode linkage method by fusion PCR is a target product end-repair process, A-tail generation (A-tailing process), bar code ligation process by enzyme, The purification process and the PCR process for amplifying the product can be omitted, which saves time and cost, and has the advantage of low error rate due to fewer steps.

Therefore, the method according to one embodiment of the present invention performs high resolution HLA typing analysis at low cost and high degree of freedom compared to the method by ligation by binding to the DNA of analysis by Fusion PCR using an adapter and a barcode sequence as a primer. Can be done.

In one embodiment of the present invention, sequencing by NGS is performed by Illumina's MiSeq, NextSeq 500, Hiseq 2000, Hiseq 2500, Hiseq 3000, Hiseq 4000, Roche's 454 FLX Titanium, GS Junior, Life Technologies' Ion Torrent PGM, Ion Proton, SOLiD3, SOLiD4 and the like can be carried out. Primers with adapters and barcodes suitable for each NGS instrument can be designed or selected.

In one embodiment of the present invention, using a next-generation sequencing, the method of performing high resolution histocombination typing analysis may further comprise the step of determining the HLA typing based on the sequence after performing the NGS. .

In one embodiment of the present invention, determining the HLA type based on the sequence determined by the NGS may be performed using software (Omixon Target HLA Typing Program: http://www.omixon.com/hla/ ).

One aspect of the present invention is a primer for amplifying HLA-A of SEQ ID NO: 1 to 6, a primer for amplifying HLA-B of SEQ ID NO: 7 to 12, and a primer for amplifying HLA-DRB1 of SEQ ID NO: 13 and 14 and SEQ ID NO: 115 to Provided is a kit for high resolution histocompatibility (HLA) type analysis using NGS, comprising a 210 barcodeing primer and an adapter primer of SEQ ID NO: 211.

In one embodiment of the invention, the kit may comprise a barcode primer that binds a barcode sequence with an adapter suitable for the NGS instrument used.

In one embodiment of the invention, the kit can be used to simultaneously perform high resolution HLA typing analysis on multiple samples.

In one embodiment of the invention, the kit may further comprise reagents necessary for performing PCR.

One aspect of the present invention provides a primer used for preparing a library for next generation sequencing (NGS), including a universal primer sequence for sequencing and a barcode sequence for sample identification.

The primers can be used to PCR-couple sequencing universal primer sequences and sample identification barcode sequences to a sample to produce a library for NGS, without limitation by the NGS instrument. The primer may be used in target-resequencing technique in addition to HLA typing.

In one embodiment of the present invention, the sequencing universal primer sequence may be SEQ ID NO: 111 or 112, but is not limited thereto. Typically primers used for sequencing can be used.

In one embodiment of the invention, the sample identification barcode sequence may be one or more of SEQ ID NO: 15 to 110.

The bar code sequence for sample identification may be designed in consideration of the number of samples to be analyzed simultaneously, and the length may be 4 to 20 bp, or more, depending on the number of samples. Barcoding allows simultaneous analysis of multiple samples, for example 96 or more.

In one embodiment of the invention, the primer may comprise an adapter sequence according to the NGS device used.

In one embodiment of the invention, the adapter sequence may be SEQ ID NO: 113 or 114 suitable for Miseq.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

The high resolution HLA-A, B, DRB1 allele type analysis method using next generation sequencing technology (NGS) according to an embodiment of the present invention solves the problem of SBT (sequence-based typing) which is a high resolution analysis method of HLA. Hematopoietic stem cell volunteers, which are suitable for use as a pre-transplantation test and require large-scale HLA analysis detection, allowing accurate results analysis without phase-ambiguity at high resolution and low cost, taking into account technical convenience. It is also possible to apply for a registration database.

1 shows the structure of HLA class I and class II genes analyzed for HLA typing according to one embodiment of the invention.

Figure 2 is a schematic diagram showing a PCR (fusion PCR) to combine the target PCR and adapter and barcode for HLA type analysis according to an embodiment of the present invention. 2A shows a PCR that binds a barcode to both ends of a target sequence, and FIG. 2B shows a PCR that binds a barcode to only one end of the target sequence.

Figure 3 is a schematic diagram showing the structure of the PCR (fusion PCR) product and the primer used for binding the target PCR product and adapter and barcode for HLA type analysis according to an embodiment of the present invention.

4 shows the results of amplifying exons 2, 3, 4 of HLA-A, exons 2, 3, 4 of HLA-B, and exons 2 of HLA-DRB1 in multiple target PCR according to an embodiment of the present invention. . Figure 4a shows the results of electrophoresis of the target multiplex PCR, Figures 4b to 4d shows the results of the bioanalyzer confirmation (Group 1: exon of HLA-A, Group 2: exon of HLA-B, Group 3: of HLA-DRB1 Exon).

Figure 5 shows the Fusion PCR results performed after pooling (pooling) the product of multiple target PCR according to an embodiment of the present invention.

Example 1 Preparation of HLA-A, HLA-B, HLA-DRB1 Specific Primers

Primers designed for HLA typing are based on the IMGT / HLA Database, Release v.3.18.0 (http://www.ebi.ac.uk/imgt/hla/, October 2014) nucleotide sequence. In A and B, HLA class II was developed to enable type analysis of DRB1 moieties and to amplify thousands of alleles.

1 depicts the gene structure of HLA classes I and II and target regions for HLA typing analysis.

The HLA alleles known to date vary widely with 2,946 HLA-A, 3,693 HLA-B, and 1,582 DRB1 (as of October 2014).

Therefore, for high resolution HLA type analysis, it is important to design primers capable of amplifying all types having different nucleotide sequences and not causing non-specific amplification. By aligning the base sequences of all types, all of the mutated portions of the base sequences were applied, and the length of primer and Tm (Melting Temperature) conditions were established so that the PCR temperature conditions could be unified. In addition, all target amplification primers were commonly assigned a universal base sequence so that an adapter including a barcode base sequence for sample identification could be linked in a subsequent PCR.

This example provides primers that can be used for high resolution HLA typing based on Illumina Miseq sequencing technology, but is not limited to devices as it is designed to modify the nucleotide sequence to suit the characteristics of personal identification barcodes and universal sequences. Do not.

The newly designed primers in this example independently amplify exons 2, 3, 4 of HLA-A, B gene, and exon 2 of HLA-DRB1 gene, and their PCR products are less than 550 bp in length (NGS instrument). Specific barcode adapter sequence may be reduced according to the length), and was prepared in consideration of the annealing temperature of the primers to enable multiplex (multiplex) PCR.

Since HLA is the most polymorphic gene found in humans, select a highly conserved sequence region to enable specific amplification of HLA-A, B, DRB1, and all of their types, namely alleles, Primers were designed using degenerate codons having two or more bases in one location.

Primer sequences for amplifying exons 2, 3, 4 of HLA-A, B and exon 2 of HLA-DRB1 prepared in this Example are shown in Table 1 below.

Table 1

Degenerate sites in primers: R = A / G, Y = C / T, M = A / C, K = G / T, S = C / G, W = A / T, H = A / C / T, B = C / G / T, V = A / C / G, D = A / G / T, N = A / C / G / T.

The underlined parts in Table 1 represent universal sequences for sequencing.

Example 2. Amplification of HLA-A, HLA-B, HLA-DRB1 Genes Using Multiplex PCR

Target PCR was performed using primers designed in Example 1 to amplify all possible types of HLA-A, B, and DRB1 for high resolution HLA typing. PCR may be performed separately for each target region, but in the present embodiment, a multiple PCR method of simultaneously amplifying a plurality of targets was used in order to reduce analysis time, reduce cost, and ease of analysis. Since primers for amplifying each target region prepared in Example 1 are designed to integrate annealing temperature and PCR conditions, multiple PCR can be performed.

DNA was extracted from 96 blood samples and each sample was named barcodes 01-96. Target PCR reactions are performed in a total of two 96 well plates, plates 1 and 2, with each 96 plate mixed primers capable of amplifying HLA-A, exon 2 of B, exon 3, exon 4 and exon 2 of DRB1. Was injected.

96 well plate 1 was injected with 10 μM of HLA-A-exon 4, HLA-B- exon 2, and 10 μM of HLA-B-exon 4 in a 3: 3: 1 mixed ratio, and in 96 well plate 2, HLA- A-exon 2, HLA-A-exon 3, HLA-B-exon 3, HLA-DRB1-exon 2 primer ratio was mixed and injected in a ratio of 1: 1: 1: 1.5.

Libraries for NGS are prepared via PCR that binds an adapter for NGS and a barcode sequence for sample identification to a product obtained by PCR amplifying the target region, and then analyzes the sequence by NGS. When preparing a library for NGS, the amount of target area should be the same or similar so that the NGS results for all target areas can be obtained with the same accuracy, so quantification of each target area is required. For the simplification of this quantification step, all target regions, namely HLA-A-Exon 4 (AE4), HLA-B-Exon 2 (BE2), HLA-B-Exon 4 (BE4), HLA-A-Exon 2 Amplification of the target region was performed so that the ratios of the amplification products of (AE2), HLA-A-exon 3 (AE3), HLA-B-exon 3 (BE3), and HLA-DRB1-exon 2 (DRB1E2) were approximately equal. The proportion of primer pairs was adjusted in PCR for.

Polymerization buffer and DNA polymerase were injected into two 96 well plates, and 1.5 μl of primer and 1 μl of sample DNA were put in the respective 96 well plates, and the amplification process using the polymerase chain reaction device was performed.

Polymerization buffer containing dNTP 0.2 mM, betaine 0.3M, 1X Phusion buffer was used, and Phusion Hot Start II High-Fidelity DNA polymerase 0.2 unit (Themo Scientific) was used as DNA polymerase.

Polymerase chain reaction conditions were performed once for 30 seconds at 98 ° C., 1 minute at 98 ° C., 30 seconds at 60 ° C., and 30 seconds at 72 ° C., and finally, 5 minutes at 72 ° C. The test was carried out once, and the PCR instrument was using Bio-Rad C1000. 4 shows the results of electrophoresis and bioanalyzer identification of HLA target multiplex PCR.

Example 3. Fusion PCR for Sample Identification Barcoding

Fusion PCR was performed to connect the sample identification barcode sequence and the adapter to the HLA target PCR product obtained in Example 2. This method combines adapters and barcodes by PCR without using expensive NGS library preparation kits, which saves time and money, and can be applied to target-resequencing techniques in addition to HLA testing. Do.

The 96 well plate 1 and 96 plate 2 amplification products amplified by the target PCR were pooled by the same sample into one plate, and a fusion PCR was performed using primers linked with 96 types of NGS-Miseq identification barcodes.

96 types of NGS-Miseq identification barcoding primers were injected into 96 well plate 3 at a concentration of 5 μM. The polymerization buffer and the polymerization enzyme were injected into the 96 well plate 3, and 1 μl of the PCR product integrated for each sample was put in each 96 well plate corresponding location, and the amplification process using the polymerase chain reaction device was performed. At this time, the complete polymer solution containing dNTP 0.2 mM, betaine 0.3M, 1X Phusion buffer, DNA polymerase Phusion Hot Start II High-Fidelity DNA polymerase 0.4 unit (Themo Scientific) was used.

At this time, the polymerase chain reaction conditions were performed once for 30 seconds at 98 ° C., 15 seconds at 98 ° C., 30 seconds at 60 ° C., and 30 seconds at 72 ° C., and finally, 5 minutes at 72 ° C. One run and the PCR instrument used Bio-Rad C1000.

2A and 2B schematically show how to perform target amplification PCR followed by Fusion PCR of HLA. In addition, Figure 3 shows the structure of the primers and PCR products used in target amplification PCR and Fusion PCR.

Barcoding primers comprising a barcode and adapter for sample identification include universal primers that are complementary to the target PCR amplification products. The identification barcode primer sequence consists of the structure of an adapter sequence-barcode sequence-universal primer sequence. The adapter sequence, the universal primer sequence, and the barcode sequence among the 96 types of NGS-Miseq identification barcoding primer sequences prepared and used in this example are shown in Tables 2 and 3, respectively. In addition, adapter sequences and universal primer sequences among the adapter primers used in pairs with barcoded primers in Fusion PCR are described in Table 4 below.

TABLE 2

TABLE 3

Table 4

Example 4 Sequencing and HLA Type Determination Using NGS

The Fusion PCR product obtained in Example 3 was purified and quantified.

First, 5 μl of PCT product taken from each of 96 wells was mixed into one 1.5 ml tube to obtain 100 μl, and 100 μl of Agencourt AMPure XP bead was mixed and attached to magnetic beads to remove primer and dNTP residue, 80 After washing the beads with% ethanol, PCR products were dissolved in 50 μl of Qiagen Elution buffer.

It was determined after the mixed sample bar code samples purified using Quantus ^TM Fluorometer (Promega, USA) apparatus. For the measurement, Quant-iT PicoGreen ^® dsDNA ^TM Reagent Kit of Quant-iT ^TM PicoGreen ^® dsDNA reagent was prepared by diluting the reagent in 1X TE 1.5 ㎕ reagent 298.5 ㎕. 200 μl blank reagent (100 μl Quant-iT ^TM PicoGreen ^® dsDNA Reagen reagent diluted in 100 μl 1X TE reagent), 200 μl standard reagent (Quant-iT ^TM diluted 2 μl Lambda DNA standard reagent in 98 μl 1X TE reagent) 100 μl of PicoGreen ^® dsDNA Reagen reagent), 200 μl of sample reagent (100 μl of Quant-iT ^™ PicoGreen ^® dsDNA Reagen reagent diluted with 1 μl of sample in 99 μl of 1X TE reagent) in 0.5 ml tubes ^It was measured in a ^TM Fluorometer. The final mixed sample, labeled barcodes 01-96, was determined to be about 30-50 nM as measured by Quantus ^™ Fluorometer.

According to the Miseq Sample Preparation Instructions, the Miseq library product prepared at 10 nM was diluted to 150 pM final concentration to obtain 540 μl, and 60 μl of 12.5 pM Phix library was added to prepare a total of 600 μl final product. Then, the sequence of each sample was determined by the Illumina Miseq program. In summary, the sequence determined by the Illumina Miseq program is output as data in the form of a fastq file, and then, after checking the quality of the determined sequence such as N base, GC content, and Q30 base fraction, the software (Omixon Target HLA Typing program) is determined based on the determined sequence. : http://www.omixon.com/hla/) was used to determine the HLA type of each sample.

The results obtained by the NGS were compared with the results by SSOP, which is a medium resolution analysis method for the same sample.

Tables 5 to 7 below show the results for HLA-A, HLA-B, and HLA-DRB1, respectively.

Table 5

1. SSOP results: homozygote or heterozygote with NGS results.

Table 6

TABLE 7

1: SSOP results: homozygote or heterozygote results with NGS results.

The comparison with SSOP shows that the analysis by NGS enables more accurate and high resolution HLA type determination.

The sequences of SEQ ID NO: 1 to SEQ ID NO: 211 described herein are shown in the attached sequence listing.

Claims (10)

1. As a method of performing high resolution histocompatibility (HLA) typing using next generation sequencing (NGS),

   Amplifying the exon regions of HLA-A, HLA-B, and HLA-DRB1 in the sample to obtain a target PCR product,

   Using the obtained target PCR product as a template, a fusion PCR using a barcode primer and an adapter primer comprising a universal primer sequence for sequencing, a barcode sequence for sample identification, and an adapter sequence was performed to perform an adapter for NGS, and Obtaining a barcoded PCR product bound to a barcode for sample identification,

   Performing NGS on the barcoded PCR product.
2. The method of claim 1, wherein amplifying the exon regions of HLA-A, HLA-B, and HLA-DRB1 to obtain a target PCR product is performed by multiplex PCR.
3. The method of claim 1, wherein the amplifying the exon region of the HLA-A, B and DRB1 to obtain a target PCR product is a primer for amplifying the HLA-A of SEQ ID NO: 1 to 6, HLA-B amplification of SEQ ID NO: 7 to 12 And a primer selected from the group consisting of primers for amplifying HLA-DRB1 of SEQ ID NOs: 13 and 14.
4. The method of claim 2, wherein the multiplex PCR amplifies exons 2, 3, 4 of HLA-A, exons 2, 3, 4 of HLA-B, and exon 2 of HLA-DRB1.
5. The method of claim 1, wherein the obtaining of the barcoded PCR product is performed using a primer selected from the group consisting of barcoded primers of SEQ ID NOs: 115 to 210 and an adapter primer of SEQ ID NO: 211.
6. Primers for amplifying HLA-A of SEQ ID NOs: 1-6, primers for amplifying HLA-B of SEQ ID NOs: 7-12, primers for amplifying HLA-DRB1 of SEQ ID NOs: 13 and 14, and barcode primers of SEQ ID NOs: 115 to 210; Kit for high resolution histocompatibility (HLA) type analysis comprising an adapter primer of SEQ ID NO: 211.
7. A primer used for fabricating a library for next generation sequencing (NGS), comprising a universal primer sequence for sequencing and a barcode sequence for sample identification.
8. The primer of claim 7, wherein the sequencing universal primer sequence is SEQ ID NO: 111 or 112. 9.
9. The primer of claim 7, wherein the sample identification barcode sequence is selected from 15 to 110. 9.
10. 10. The primer of any one of claims 7-9, wherein the primer further comprises an adapter sequence according to the NGS device used.

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