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WO2016129759A1 - Procédé et kit d'analyse par typage hla à haute efficacité, haute résolution basé sur une technique de séquençage de nouvelle génération - Google Patents

Procédé et kit d'analyse par typage hla à haute efficacité, haute résolution basé sur une technique de séquençage de nouvelle génération Download PDF

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Publication number
WO2016129759A1
WO2016129759A1 PCT/KR2015/006773 KR2015006773W WO2016129759A1 WO 2016129759 A1 WO2016129759 A1 WO 2016129759A1 KR 2015006773 W KR2015006773 W KR 2015006773W WO 2016129759 A1 WO2016129759 A1 WO 2016129759A1
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WIPO (PCT)
Prior art keywords
hla
primer
sequence
pcr
ngs
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Ceased
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PCT/KR2015/006773
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English (en)
Korean (ko)
Inventor
조대연
신인경
문서윤
최세림
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LABGENOMICS CO Ltd
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LABGENOMICS CO Ltd
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Publication date
Application filed by LABGENOMICS CO Ltd filed Critical LABGENOMICS CO Ltd
Publication of WO2016129759A1 publication Critical patent/WO2016129759A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • 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.
  • SBT sequence-based typing
  • SBT sequence-based typing
  • an aspect of the present invention is a method for performing high resolution histocompatibility (HLA) type analysis using next-generation sequencing (NGS),
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • the barcode sequence acts as a label to identify each sample, allowing multiple barcoded samples to be integrated and analyzed simultaneously.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • Tables 5 to 7 below show the results for HLA-A, HLA-B, and HLA-DRB1, respectively.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Evolutionary Biology (AREA)
  • Medical Informatics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Theoretical Computer Science (AREA)
  • Cell Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé et un kit qui permettent de simultanément doser le typage des allèles HLA-A, B, et DRB1 par analyse à haute résolution à l'aide d'une technique de séquençage de nouvelle génération (NGS).
PCT/KR2015/006773 2015-02-11 2015-07-01 Procédé et kit d'analyse par typage hla à haute efficacité, haute résolution basé sur une technique de séquençage de nouvelle génération Ceased WO2016129759A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020150020961A KR101782806B1 (ko) 2015-02-11 2015-02-11 차세대염기서열분석기술 기반의 고효율, 고해상도 조직적합성 형별 분석 방법 및 키트
KR10-2015-0020961 2015-02-11

Publications (1)

Publication Number Publication Date
WO2016129759A1 true WO2016129759A1 (fr) 2016-08-18

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WO (1) WO2016129759A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102131293B1 (ko) 2018-06-29 2020-07-07 경북대학교 산학협력단 서열-특이적 올리고뉴클레오티드 프로브 방법에 의한 인간백혈구항원 형별 검사에서 인간백혈구항원의 최종 형별 판정 방법

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009049889A1 (fr) * 2007-10-16 2009-04-23 Roche Diagnostics Gmbh Génotypage hla à haute résolution et haut débit par séquençage clonal
KR20130142523A (ko) * 2012-06-19 2013-12-30 (주)지노첵 차세대 염기서열 분석법을 위한 융합 프라이머의 설계방법 그리고 이러한 융합 프라이머 및 차세대 염기서열 분석법을 이용한 표적 유전자의 유전자형 분석방법
US20140206005A1 (en) * 2011-07-21 2014-07-24 Genodive Pharma Inc. Method and Kit for DNA Typing of HLA Gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009049889A1 (fr) * 2007-10-16 2009-04-23 Roche Diagnostics Gmbh Génotypage hla à haute résolution et haut débit par séquençage clonal
US20140206005A1 (en) * 2011-07-21 2014-07-24 Genodive Pharma Inc. Method and Kit for DNA Typing of HLA Gene
KR20130142523A (ko) * 2012-06-19 2013-12-30 (주)지노첵 차세대 염기서열 분석법을 위한 융합 프라이머의 설계방법 그리고 이러한 융합 프라이머 및 차세대 염기서열 분석법을 이용한 표적 유전자의 유전자형 분석방법

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GRUMBT ET AL.: "Diagnostic Applications of Next Generation Sequencing in Immunogenetics and Molecular Oncology", TRANSFUSION MEDICINE AND HEMOTHERAPY, vol. 40, no. 3, 2013, pages 196 - 206 *
LANGE ET AL.: "Cost-Efficient High-Throughput HLA Typing by MiSeq Amplicon Sequencing", BMC GENOMICS, vol. 15, no. 63, 2014, pages 1 - 11 *

Also Published As

Publication number Publication date
KR20160098838A (ko) 2016-08-19
KR101782806B1 (ko) 2017-09-28

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