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1. CN111334868 - Construction method of novel coronavirus whole-genome high-throughput sequencing library, and kit for library construction

Office China
Application Number 202010225821.0
Application Date 26.03.2020
Publication Number 111334868
Publication Date 26.06.2020
Publication Kind A
IPC
C40B 50/08
CCHEMISTRY; METALLURGY
40COMBINATORIAL TECHNOLOGY
BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES, IN SILICO LIBRARIES
50Methods of creating libraries, e.g. combinatorial synthesis
08Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
C12Q 1/70
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING 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
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
70involving virus or bacteriophage
C12Q 1/6869
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING 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
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
68involving nucleic acids
6869Methods for sequencing
G16B 20/20
GPHYSICS
16INFORMATION AND COMMUNICATION TECHNOLOGY SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
20ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
20Allele or variant detection, e.g. single nucleotide polymorphism detection
G16B 20/30
GPHYSICS
16INFORMATION AND COMMUNICATION TECHNOLOGY SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
20ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
30Detection of binding sites or motifs
C12R 1/93
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C-C12Q75
1Microorganisms
92Viruses
93Animal viruses
CPC
C12Q 1/6869
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
68involving nucleic acids
6869Methods for sequencing
C12Q 1/701
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
70involving virus or bacteriophage
701Specific hybridization probes
C40B 50/08
CCHEMISTRY; METALLURGY
40COMBINATORIAL TECHNOLOGY
BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES, IN SILICO LIBRARIES
50Methods of creating libraries, e.g. combinatorial synthesis
08Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
G16B 20/20
GPHYSICS
16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
20ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
20Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
G16B 20/30
GPHYSICS
16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
20ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
30Detection of binding sites or motifs
Applicants FUZHOU FURUI MEDICAL INSPECTION LABORATORY CO., LTD.
福州福瑞医学检验实验室有限公司
CHINESE ACADEMY OF MEDICAL SCIENCES
中国医学科学院
Inventors WANG YANG
王洋
LI JIE
李杰
WANG CHEN
王辰
GAO HANLIN
高汉林
GUO CHAO
郭超
WANG JIANWEI
王健伟
REN LILI
任丽丽
YANG MING
杨明
LIU JING
刘静
ZHAO YE
赵晔
Agents 北京路浩知识产权代理有限公司 11002
Title
(EN) Construction method of novel coronavirus whole-genome high-throughput sequencing library, and kit for library construction
(ZH) 新型冠状病毒全基因组高通量测序文库的构建方法以及用于文库构建的试剂盒
Abstract
(EN)
The invention provides a construction method of a novel coronavirus whole-genome high-throughput sequencing library, and a kit for library construction. The method comprises the following steps: 1) carrying out reverse transcription on virus RNA; (2) carrying out a first round of PCR reaction by utilizing multiple amplification primers of an anchoring part Illumina linker sequence; and 3) carryingout a second round of PCR reaction by using a labeled Illumina library amplification primer, and purifying the amplification product to obtain the high-throughput sequencing library. The anchoring multiplex amplification primer combination provided by the invention is utilized, so the efficient targeted enrichment can be performed on the genome of the novel coronavirus COVID-19, the influence that an existing method is low in targeting, low in experimental timeliness and prone to being brought into host background pollution is overcome, COVID-19 virus whole genome sequencing can be completedwithin a short time with the small sequencing data volume and cost, so differential diagnosis and virus mutation identification of the COVID-19 virus are achieved.

(ZH)
本发明提供新型冠状病毒全基因组高通量测序文库的构建方法以及用于文库构建的试剂盒。所述方法包括:1)病毒RNA反转录;2)利用锚定部分Illumina接头序列的多重扩增引物进行第一轮PCR反应;3)利用标签化Illumina文库扩增引物进行第二轮PCR反应,扩增产物经纯化,得到高通量测序文库。利用本发明提供的锚定多重扩增引物组合,能够对新型冠状病毒COVID‑19的基因组进行高效靶向富集,克服现有方法靶向性低,实验时效低,且易带入宿主背景污染的影响,有利于在较少的测序数据量、成本下,较短时间内完成COVID‑19的病毒全基因组测序,从而实现COVID‑19病毒的鉴别诊断以及病毒突变鉴定。