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WO2017222164A1 - Library primer set for sequencing and method for preparing library - Google Patents

Library primer set for sequencing and method for preparing library Download PDF

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Publication number
WO2017222164A1
WO2017222164A1 PCT/KR2017/004749 KR2017004749W WO2017222164A1 WO 2017222164 A1 WO2017222164 A1 WO 2017222164A1 KR 2017004749 W KR2017004749 W KR 2017004749W WO 2017222164 A1 WO2017222164 A1 WO 2017222164A1
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sequence
sequencing
adapter
primer
library
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French (fr)
Korean (ko)
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김세일
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Korea Research Institute of Standards and Science
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Korea Research Institute of Standards and Science
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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
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • 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
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • 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

  • the present invention relates to a primer set for a library including a fusion primer for gene amplification for sequencing and a method for preparing the library, and more specifically, to a sequencing library of a target gene to be analyzed through next-generation sequencing. It relates to a primer set used to prepare through a simplified process and a library manufacturing method using the same.
  • NGS Next generation sequencing
  • Next-generation sequencing analyzers such as Mysq, Hiseq, and Nextseq from Illuminaa, and Sequel next-generation sequencing and life technologies from Pacific Bioscience Next-generation sequencing analyzers such as Ion PGM and Ion Proton from Life Technologies have been developed and used.
  • a sequence of processes such that a genome, a transcript, or a target gene can be used in a next-generation sequencing analyzer is called a sequencing library preparation.
  • the sequencing library of Illumina's sequencing analyzer can amplify specific target genes through polymerase chain reaction (PCR), purify amplified genes, and purify them.
  • 2 Adaptation of the adapter sequence, purification of the labeled base sequence, and the gene combining the adapter sequence (16S Metagenomic Sequencing Library Preparation, Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System, Part # 15044223 Rev. B).
  • the process can take 6.5 to 10 hours or less than one day if automated.
  • loss and distortion of the target gene occurs during each process, so minimizing it is necessary to obtain accurate sequence information.
  • the sequencing library produced by BIOO scientific takes relatively less time than the Illumina method, but requires two chain polymerase reactions and two purification processes (NEXTflex TM Metagenomics Application for Sciclone NGS Workstation, (Compatible with NEXTflex TM 16S V1-V3 Amplicon-Seq Kit, NEXTflex TM 16S V4 Amplicon-Seq Kit 2.0, and NEXTflex TM 18S ITS Amplicon-Seq Kit) ⁇ Bioo Scientific Corp. 2016 V16.02).
  • the prepared sequencing library is quantitatively determined and is the target of the next generation sequencing analyzer with the optimal amount required for sequencing.
  • next-generation sequencing analysis on specific target genes is microbial ecology.
  • the technology is being used in many fields such as analysis.
  • diseases related to nutrients such as obesity
  • diseases that were not considered to be directly related to microorganisms such as diabetes and autoimmune diseases
  • the microbial ecology study mainly uses 16S rRNA gene, which is a taxonomic indicator gene.
  • Roche's next-generation sequencing analyzer whose main principle is pyrosequencing, was used, but the analyzer will be discontinued in 2016.
  • Such microbial community analysis is to estimate the composition ratio of a specific microorganism in a sample by obtaining the base sequence of the indicator gene and comparing the specific microorganism-derived index gene base sequence to the total number of obtained base sequence. This is because the analysis is performed on the assumption that the ratio of microorganisms in the actual sample and the composition ratio of the amplified microorganism-derived indicator genes through the chain polymerase reaction is the same, or similar. Should be minimized. Such distortion is known to occur during the chain polymerase reaction, the purification of the sample.
  • Roche's sequencing library was manufactured and used to minimize such distortion and to produce a sequencing library at a simple and high speed.
  • the sequencing library of Roche's analyzer was amplified by an emulsion-PCR (em-PCR).
  • the Illumina analyzer is amplified by the bridge chain polymerase reaction because both primers are fixed in the flow cell, and the sequencing principle is different. Therefore, Roche's fusion primer for next-generation sequencing analyzer can be applied to the next-generation sequencing analyzer of Illumina. none. Therefore, as described above, in order to manufacture a sequencing library of amplified genes for the next generation analyzer of Illumina, it is essential to attach an adapter capable of complementarily binding primers fixed to the flow cell to the amplified genes. need. For this reason, currently used sequencing library manufacturing method is time-consuming and intermediate process is difficult to perform the non-skilled person as well as the risk of distortion or loss of the sample, the situation is urgently required to solve this problem.
  • the present invention has been made to solve the above problems, by minimizing the intermediate process to minimize the loss and distortion of the sample and to prepare a sequencing library simple, fast fusion primers for gene amplification for sequencing It is an object to provide a primer set for a library to be included.
  • another object of the present invention is to provide a method for producing a sequencing library of a target gene to be analyzed through a next generation sequencing through a simplified process at high speed.
  • the primer set for the library for sequencing of the present invention is
  • the first forward adapter that binds to the front oligo immobilized on the flow cell, the second forward adapter that binds to the forward sequencing primer, and the forward gene specific sequence that binds to the forward gene of the target gene are listed in order.
  • Front primer comprising;
  • the first rear adapter that binds to the rear oligo immobilized on the flow cell, the second rear adapter that binds to the rear sequencing primer, and the rear gene specific sequence that binds to the rear gene of the target gene It characterized in that it comprises a rear primer to include.
  • primer set for the library for sequencing of the present invention may further include a front mixed space sequence for improving sequence diversity between the second front adapter and the front gene specific sequence.
  • primer set for the library for sequencing of the present invention may further include a rear mixing space sequence for improving sequence diversity between the second rear adapter and the rear gene specific sequence.
  • the primer set for the library for sequencing of the present invention is between the first anterior adapter and anterior gene specific sequence, specifically between the first anterior adapter and the second anterior adapter, between the second anterior adapter and anterior mixed spatial sequence
  • an anterior marker recognition sequence may be further included to facilitate identification of the origin of the sample.
  • the primer set for the library for sequencing of the present invention is between the first rear adapter and the rear gene specific sequence, specifically between the first rear adapter and the second rear adapter, between the second rear adapter and the rear mixed spatial sequence
  • a back label recognition sequence may be further included to facilitate identification of the origin of the sample.
  • primer set for the library for sequencing of the present invention may further comprise a front linker between the front mixing space sequence and the front gene specific sequence.
  • primer set for the library for sequencing of the present invention may further include a rear linker between the rear mixing space sequence and the rear gene specific sequence.
  • the first forward adapter may be the sequence of SEQ ID NO: 1.
  • the first rear adapter may also be the sequence of SEQ ID NO: 2.
  • front label recognition sequence may be any one of SEQ ID NO: 3 to 10.
  • the back label recognition sequence may be any one of SEQ ID NO: 11 to 22.
  • the base constituting the front mixed space sequence is absent or 1 to 23, and the base constituting the rear mixed space sequence is absent or 1 to 15, or
  • the base constituting the front mixed space sequence may be absent or 1 to 15, and the base constituting the rear mixed space sequence may be absent or 1 to 23.
  • A a first anterior adapter that binds an anterior oligo immobilized to a flow cell, a second anterior adapter that binds an anterior sequencing primer, and an anterior gene specific sequence that binds an anterior gene of a target gene
  • a front primer comprising in the order listed
  • (C) characterized in that it comprises the step of purifying the amplified base sequence.
  • the method for preparing a sequencing library of the present invention may further include a front mixed space sequence for improving sequence diversity between the second front adapter and the front gene specific sequence.
  • the method for preparing a sequencing library of the present invention may further include a rear mixing space sequence for improving sequence diversity between the second rear adapter and the rear gene specific sequence.
  • the method for preparing a sequencing library of the present invention includes a first anterior adapter and an anterior gene specific sequence, specifically, between a first anterior adapter and a second anterior adapter, between a second anterior adapter and an anterior mixed spatial sequence, And at a position selected from the group consisting of an anterior mixed space sequence and an anterior gene specific sequence, an anterior label recognition sequence for facilitating identification of a sample.
  • the method for preparing a sequencing library of the present invention is provided between a first rear adapter and a rear gene specific sequence, specifically, between a first rear adapter and a second rear adapter, between a second rear adapter and a rear mixed spatial sequence, And at a position selected from the group consisting of a posterior mixing space sequence and a posterior gene specific sequence, a rear labeling sequence that facilitates identification of the origin of the sample.
  • the method for preparing a sequencing library of the present invention may include only one amplification step and one purification step.
  • the sequencing library manufacturing method of the present invention it is possible to perform the chain polymerase reaction and the nucleic acid purification process that had to be performed two or more times only once. As a result, the library can be prepared in about 2 hours including the time for amplifying the template.
  • sequencing library prepared by the present invention can be used not only for the next-generation sequencing analyzer of Illumina Corporation, but also for analyzers using the same principle.
  • 1 is a conceptual diagram illustrating a manufacturing process of a sequencing library using a conventional primer.
  • Figure 2 is a conceptual diagram showing an embodiment of the manufacturing process of the sequencing library using the fusion primer of the present invention.
  • the fusion primer consists of a front primer and a back primer, each primer again to the first adapter (170, 270) that binds to the oligo immobilized in the flow cell, to facilitate the origin of the sample Label recognition sequences (160, 260), second adapters (150, 250) to bind to sequencing primers, mixed spatial sequences (140, 240) to enhance sequence diversity, gene specific sequences (120) to bind to template (10) , 220), linker (130, 230) for connecting the gene-specific nucleotide sequence (120, 220) with the rest of the sequence.
  • the linkers 130 and 230 are nucleotide sequences connecting the target gene specific nucleotide sequences and other portions thereof, and may have various configurations or lengths of the nucleotide sequences.
  • the primer set for the library for sequencing of the present invention includes a front primer and a back primer, each primer is a first adapter (170, 270), a second adapter (150, 250), and gene specific The enemy sequences 120, 220 are included in the order listed.
  • each of the primers has a mixed spatial sequence (140, 240) between the second adapter (150, 250) and the gene specific sequence (120, 220), and the first adapter (170, 270) and gene specific Labeling sequences 160 and 260 between the sequences 120 and 220 and also linkers 130 and 230 between the gene specific sequences 120 and 220 and the remaining sequences.
  • the greatest feature of the present invention is to reduce the number of repetitions of the chain polymerase reaction and purification process by collecting and fusing sequences having different roles in one primer and then binding them to both ends of the template (10). This reduction in the number of iterations not only shortens the time required to produce the sequencing library, but also prevents distortion or loss of the sample and enables the skilled person to produce the library skillfully.
  • the first adapters 170 and 270 bind to oligos, i.e., oligonucleotides, immobilized in a flow cell for amplification.
  • the present invention includes, as such first adapters 170 and 270, a first front adapter 170 located in front of the mold 10 and a first rear adapter 270 located behind the mold 10.
  • the first front adapter 170 of the present invention may be a sequence of SEQ ID NO: 1
  • the first rear adapter 270 may be a sequence of SEQ ID NO: 2.
  • the second adapter 150, 250 serves to bind the sequencing primer for sequencing, and thus may be modified to have a complementary sequence for the sequencing primer used.
  • the second front adapter 150 of the present invention binds to the front sequencing primer, and the second back adapter 250 binds to the back sequencing primer.
  • gene specific sequences 120 and 220 may bind to both terminal genes of the template 10, and the sequence may be changed according to the template 10.
  • the nucleotide sequence is not interrupted. Without killing it is possible to analyze at once.
  • the present invention may further include a mixed spatial sequence (140, 240) between the second adapter (150, 250) and the gene specific sequence (120, 220).
  • the mixed spatial sequences 140 and 240 serve to prevent a sudden drop in the accuracy of analysis when the sequences to be analyzed are similar to each other and thus have low diversity.
  • the base constituting the front mixed space sequence 140 or the rear mixed space sequence 240 is (condition 1) the base constituting the front mixed space sequence 140 is absent or 1 to 23, and the rear mixed space sequence 240 ) Base is absent or is 1 to 15, or (condition 2) bases constituting the front mixed space sequence 140 is absent or 1 to 15 bases, and bases constituting the rear mixed space sequence 240 is absent Or to satisfy 1 to 23. Due to the presence of such mixed spatial sequences (140, 240) it is possible to significantly increase the accuracy of the analysis when the diversity between the sequences, such as the microbial population described above is low.
  • the rear mixed space sequence 140 is mixed between the second anterior adapter 150 and the anterior gene specific sequence 120, and the rear mixed between the second rear adapter 250 and the rear gene specific sequence 220.
  • the spatial sequence 240 may further include.
  • the present invention may further include a label recognition sequence (160, 260) between the first adapter (170, 270) and the gene specific sequence (120, 220), the label recognition sequence (160, 260) is analyzed It serves to facilitate identification of which sample the sequence is being derived from.
  • forward mixing between the first anterior adapter 170 and the anterior gene specific sequence 120, specifically, between the first anterior adapter 170 and the second anterior adapter 150 is forward mixed with the second anterior adapter 150.
  • the front label recognition sequence 160 may be any one of SEQ ID NOs: 3 to 10
  • the rear label identification sequence 260 may be any one of SEQ ID NOs: 11 to 22.
  • the invention may further link gene specific sequences 120 and 220 with the remaining sequences via linkers 130 and 230.
  • the front linker 130 between the front mixed space sequence 140 and the front gene specific sequence 120, and the rear linker 230 between the rear mixed space sequence 240 and the rear gene specific sequence 220. ) May be further included.
  • (C) characterized in that it comprises the step of purifying the amplified base sequence.
  • the method for preparing a sequencing library of the present invention may include only the amplifying and purifying steps only once, unlike the prior arts. That is, according to the present invention, the sequencing primer can be used to kill sequencing at once and complete at once without interrupting sequencing.
  • Example described below was to determine the population composition ratio through 16S rRNA gene-based cluster analysis in the microbial community genome.
  • the microbial chromosome in the sample was extracted from the sample collected using soil microbial dielectric kit (MO BIO, USA). Using the extracted chromosome as a template, 16S rRNA gene was amplified by PCR. 16S rRNA genes were amplified using bac27f (AGAGTTTGATCMTGGCTCAG) and bac1492r (GGTTACCTTGTTACGACTT) commonly used in various taxa as 16S rRNA specific primers. The amplified gene was conjugated to the pGEM-T easy vector (Promega, USA) to transform E. coli to perform cloning. Plasmid DNA was extracted from the recombinant E. coli obtained by cloning using the Qiagen plasmid mini kit (Qiagen, Germany). The obtained plasmid was quantified by Sybr Green qPCR using pGEM-T easy vector specific primers.
  • the chain polymerization reaction conditions are shown in Table 1. Sequence information of the fusion primers are shown in Table 2, Table 3, Table 4. Differences from the sequencing library manufacturing process using conventional primers can be seen from FIGS. 1 and 2.
  • amplified product was purified using AMPure XP beads (Agencourt, France), quantified by qPCR and pooled each purified product was used as a sequencing library.
  • Test Example Securing and analyzing nucleotide sequences through next-generation sequencing
  • nucleotide sequence information was obtained using a 300bp PE (v3) sequencing kit in Mythal (Illumina, USA).
  • the fusion primers can be used for academic research or quality control using microbial community analysis or to measure the relative amounts of specific genes in a sample.
  • SEQ ID NO: 04 AAGGAGTA
  • SEQ ID NO: 12 TTCTGCCT
  • SEQ ID NO: 14 TCCTCTAC
  • SEQ ID NO: 16 TGCCTCTT
  • SEQ ID NO: 17 TCGCCTTA
  • SEQ ID NO: 18 AGCGTAGC

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Abstract

The present invention relates to: a library primer set for sequencing, comprising fusion primers for amplifying genes; and a method for preparing a library, and more particularly, to: a primer set used in preparing, through a simplified process at high-speed, a sequencing library of target genes to be analyzed through next generation sequencing; and a method for preparing a library by using the same.

Description

염기서열분석을 위한 라이브러리용 프라이머 세트 및 라이브러리 제조방법Primer set and library preparation method for sequencing

본 발명은 염기서열분석을 위한 유전자 증폭용 융합 프라이머를 포함하는 라이브러리용 프라이머 세트 및 상기 라이브러리의 제조방법에 관한 것으로서, 보다 구체적으로는 차세대 염기서열 분석을 통해 분석하고자 하는 목표 유전자의 시퀀싱 라이브러리를 고속으로 간소화된 과정을 통해 제조하는 데 사용되는 프라이머 세트 및 이를 이용한 라이브러리 제조방법에 관한 것이다.The present invention relates to a primer set for a library including a fusion primer for gene amplification for sequencing and a method for preparing the library, and more specifically, to a sequencing library of a target gene to be analyzed through next-generation sequencing. It relates to a primer set used to prepare through a simplified process and a library manufacturing method using the same.

차세대 염기서열 분석기술(next generation sequencing: NGS)은 수백만에서 수천만 이상의 대량의 염기서열을 한 번에 획득하는 기술로서 유전체 연구, 미생물 다양성 연구 등 여러 분야에서 사용되고 있다. Next generation sequencing (NGS) is a technique for acquiring millions of millions or more of nucleotide sequences at once and is used in various fields such as genome research and microbial diversity research.

현재 일루미나(Illumina)사의 마이씩(Miseq), 하이씩(Hiseq), 넥스트씩(Nextseq)등의 차세대 염기서열 분석기와 퍼시픽바이오사이언스(Pacific bioscience)사의 시퀄(Sequel) 차세대 염기서열 분석기 및 라이프테크놀러지스(Life technologies)사의 아이온 피쥐엠(Ion PGM), 아이온 프로톤(Ion Proton)등 차세대 염기서열 분석기가 개발되어 사용되고 있다. Next-generation sequencing analyzers such as Mysq, Hiseq, and Nextseq from Illuminaa, and Sequel next-generation sequencing and life technologies from Pacific Bioscience Next-generation sequencing analyzers such as Ion PGM and Ion Proton from Life Technologies have been developed and used.

이러한 차세대 염기서열 분석을 통해 염기서열정보를 획득하기 위해서는 유전체, 전사체 혹은 목표 유전자가 차세대 염기서열 분석기에 사용될 수 있도록 일련의 과정을 거쳐야 하는데 이러한 과정을 시퀀싱 라이브러리 준비 (sequencing library preparation)라고 한다. In order to obtain sequencing information through such next-generation sequencing, a sequence of processes such that a genome, a transcript, or a target gene can be used in a next-generation sequencing analyzer is called a sequencing library preparation.

일루미나사의 염기서열 분석기의 시퀀싱 라이브러리는 특정 목표 유전자의 염기서열을 얻고자 하는 경우 연쇄중합효소반응(polymerase chain reaction: PCR)을 통한 특정 목표 유전자의 증폭, 증폭된 유전자(amplicon)의 정제, 정제된 유전자에 특정 시료에서 증폭된 것임을 표지할 수 표지염기서열과 플로우셀 (flow cell)에 결합되어 있는 프라이머에 상보적으로 결합하는 제 1 어댑터 및 차세대 염기서열 분석기용 시퀀싱 프라이머와 상보적으로 결합하는 제 2 어댑터 염기서열을 붙여주는 과정, 표지염기서열 및 어댑터 염기서열이 결합된 유전자의 정제과정을 거치게 된다 (16S Metagenomic Sequencing Library Preparation, Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System, Part # 15044223 Rev. B). 일루미나사의 설명에 의하면 이러한 과정은 자동화를 통할 경우 6.5 시간에서 10 시간 혹은 하루 이내의 시간이 소요된다. 또한 각 과정을 진행하는 동안 목표 유전자의 손실 및 왜곡이 발생하므로 가능한 이를 최소화하는 것이 정확한 염기서열정보를 얻는데 필요하다. The sequencing library of Illumina's sequencing analyzer can amplify specific target genes through polymerase chain reaction (PCR), purify amplified genes, and purify them. A first adapter that complementarily binds to a labeled base sequence and a primer bound to a flow cell, and an agent that complements to a sequencing primer for next-generation sequencing analyzers. 2 Adaptation of the adapter sequence, purification of the labeled base sequence, and the gene combining the adapter sequence (16S Metagenomic Sequencing Library Preparation, Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System, Part # 15044223 Rev. B). According to Illumina, the process can take 6.5 to 10 hours or less than one day if automated. In addition, loss and distortion of the target gene occurs during each process, so minimizing it is necessary to obtain accurate sequence information.

바이우 사이언티픽(BIOO scientific)에서 출시한 시퀀싱 라이브러리 제조법은 일루미나사의 제조법보다 상대적으로 적은 시간이 소모되나 두 번의 연쇄중합효소반응과 두 번의 정제과정이 필요하다 (NEXTflex™ Metagenomics Application for Sciclone NGS Workstation, (Compatible with NEXTflex™ 16S V1-V3 Amplicon-Seq Kit, NEXTflex™ 16S V4 Amplicon-Seq Kit 2.0, and NEXTflex™ 18S ITS Amplicon-Seq Kit) ⓒBioo Scientific Corp. 2016 V16.02).The sequencing library produced by BIOO scientific takes relatively less time than the Illumina method, but requires two chain polymerase reactions and two purification processes (NEXTflex ™ Metagenomics Application for Sciclone NGS Workstation, (Compatible with NEXTflex ™ 16S V1-V3 Amplicon-Seq Kit, NEXTflex ™ 16S V4 Amplicon-Seq Kit 2.0, and NEXTflex ™ 18S ITS Amplicon-Seq Kit) © Bioo Scientific Corp. 2016 V16.02).

준비된 시퀀싱 라이브러리는 정량과정을 통하여 그 양이 측정되며 염기서열 분석에 필요한 최적의 양으로 차세대 염기서열 분석기의 분석대상이 된다.The prepared sequencing library is quantitatively determined and is the target of the next generation sequencing analyzer with the optimal amount required for sequencing.

한편, 특정 목표 유전자를 대상으로 차세대 염기서열 분석을 하는 대표적인 분야 중 하나는 미생물 생태학으로써 최근에 다양한 질병과 관련이 있는 것으로 밝혀진 인간 미생물군집체 분석이나 자연환경이나 미생물 이용 하수처리장 등의 미생물군집 구조 분석등 여러 분야에서 해당 기술이 활용되고 있다. 비만과 같은 영양성분과 관련된 질환을 포함하여 당뇨병, 자가면역질환 등 미생물과 직접 관계가 없을 것이라고 여겨졌던 질병들도 미생물 군집과 연관이 있음이 알려지게 되었고 이러한 중요성에 주목한 선진국에서는 인간 미생물 군집체 프로젝트 (Human Microbiome Project) 등 다양한 연구를 진행하고 있다. 이러한 미생물 생태 연구 시 주로 분류학적 지표 유전자인 16S rRNA 유전자를 활용하게 되는데 기존에는 파이로시퀀싱(pyrosequencing)을 주 원리로 하는 로슈사의 차세대 염기서열 분석기를 주로 활용하였으나 해당 분석기는 2016년에 단종이 예정되어 있어 현재는 대부분 디엔에이 클러스터 (DNA cluster) 혹은 디엔에이 콜로니 (DNA colony)를 주 원리로 하는 일루미나사의 마이씩 기종으로 분석이 진행되고 있다. 이러한 미생물 군집 분석은 지표 유전자의 염기서열을 획득하여 전체 획득 염기서열 숫자 대비 특정 미생물 유래 지표 유전자 염기서열을 비교하여 시료 내의 특정 미생물의 구성 비율을 추정하는 것이다. 이는 실제 시료 내의 미생물 구성 비율과 연쇄중합효소반응을 통한 증폭된 미생물 유래 지표 유전자의 구성비 사이가 동일하거나 유사하다는 것을 전제로 분석이 진행되는 것이기 때문에 시료내 실제 미생물과 증폭된 미생물의 지표 유전자간의 왜곡이 최소화되어야 한다. 이러한 왜곡은 연쇄중합효소반응, 시료의 정제과정 등에서 발생하는 것으로 알려져 있다. 과거에는 이러한 왜곡을 최소화하고 간편하고 고속으로 시퀀싱 라이브러리를 제조하기 위하여 로슈사 분석기용 융합프라이머를 제조하여 사용하였으나 로슈사 분석기의 시퀀싱 라이브러리는 에멀전 연쇄중합효소반응(emulsion-PCR: em-PCR)을 통해 증폭되는 반면 일루미나의 분석기는 플로우셀에 양쪽 프라이머가 고정되어 있어서 브릿지 연쇄중합효소반응으로 증폭되며 염기서열 분석원리도 다르기 때문에 로슈의 차세대 염기서열 분석기용 융합 프라이머를 일루미나의 차세대 염기서열 분석기에 적용할 수 없다. 따라서 전술한 바와 같이 현재는 일루미나의 차세대분석기용으로 증폭된 유전자의 시퀀싱 라이브러리를 제조하기 위해서는 증폭된 유전자에 추가적으로 플로우셀에 고정된 프라이머와 상보적으로 결합할 수 있는 어댑터를 붙여주는 과정이 필수적으로 필요하다. 이 때문에 현재 사용되고 있는 시퀀싱 라이브러리 제조방법은 많은 시간이 소모되고 중간과정이 많아 비숙련자의 시행이 어려울 뿐만 아니라 시료의 왜곡이나 손실 위험이 커, 이에 대한 해결이 절실히 요구되고 있는 실정이다.On the other hand, one of the representative fields of next-generation sequencing analysis on specific target genes is microbial ecology. The technology is being used in many fields such as analysis. In addition to diseases related to nutrients such as obesity, diseases that were not considered to be directly related to microorganisms, such as diabetes and autoimmune diseases, are also known to be associated with microbial community. He is currently working on a variety of projects, including the Human Microbiome Project. The microbial ecology study mainly uses 16S rRNA gene, which is a taxonomic indicator gene. Previously, Roche's next-generation sequencing analyzer, whose main principle is pyrosequencing, was used, but the analyzer will be discontinued in 2016. Currently, most analyzes are carried out by Mya type of Illumina company, which mainly uses DNA cluster or DNA colony. Such microbial community analysis is to estimate the composition ratio of a specific microorganism in a sample by obtaining the base sequence of the indicator gene and comparing the specific microorganism-derived index gene base sequence to the total number of obtained base sequence. This is because the analysis is performed on the assumption that the ratio of microorganisms in the actual sample and the composition ratio of the amplified microorganism-derived indicator genes through the chain polymerase reaction is the same, or similar. Should be minimized. Such distortion is known to occur during the chain polymerase reaction, the purification of the sample. In the past, Roche's sequencing library was manufactured and used to minimize such distortion and to produce a sequencing library at a simple and high speed. However, the sequencing library of Roche's analyzer was amplified by an emulsion-PCR (em-PCR). On the other hand, the Illumina analyzer is amplified by the bridge chain polymerase reaction because both primers are fixed in the flow cell, and the sequencing principle is different. Therefore, Roche's fusion primer for next-generation sequencing analyzer can be applied to the next-generation sequencing analyzer of Illumina. none. Therefore, as described above, in order to manufacture a sequencing library of amplified genes for the next generation analyzer of Illumina, it is essential to attach an adapter capable of complementarily binding primers fixed to the flow cell to the amplified genes. need. For this reason, currently used sequencing library manufacturing method is time-consuming and intermediate process is difficult to perform the non-skilled person as well as the risk of distortion or loss of the sample, the situation is urgently required to solve this problem.

본 발명은 상기와 같은 문제점을 해결하기 위하여 안출된 것으로서, 중간과정을 최소화하여 시료의 손실과 왜곡을 최소화하고 간단하고 빠르게 시퀀싱 라이브러리를 제조할 수 있도록, 염기서열분석을 위한 유전자 증폭용 융합 프라이머를 포함하는 라이브러리용 프라이머 세트를 제공하는 것을 그 목적으로 한다.The present invention has been made to solve the above problems, by minimizing the intermediate process to minimize the loss and distortion of the sample and to prepare a sequencing library simple, fast fusion primers for gene amplification for sequencing It is an object to provide a primer set for a library to be included.

또한, 본 발명은 차세대 염기서열 분석을 통해 분석하고자 하는 목표 유전자의 시퀀싱 라이브러리를 고속으로 간소화된 과정을 통해 제조하는 제조방법을 제공하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to provide a method for producing a sequencing library of a target gene to be analyzed through a next generation sequencing through a simplified process at high speed.

상술한 바와 같은 목적을 달성하기 위하여, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 In order to achieve the object as described above, the primer set for the library for sequencing of the present invention is

플로우셀 (flow cell)에 고정된 전방 올리고(oligo)와 결합하는 제 1 전방 어댑터, 전방 시퀀싱 프라이머와 결합하는 제 2 전방 어댑터, 및 목표 유전자의 전방 유전자와 결합하는 전방 유전자 특이적 서열을 나열된 순으로 포함하는 전방 프라이머; 및 The first forward adapter that binds to the front oligo immobilized on the flow cell, the second forward adapter that binds to the forward sequencing primer, and the forward gene specific sequence that binds to the forward gene of the target gene are listed in order. Front primer comprising; And

플로우셀 (flow cell)에 고정된 후방 올리고(oligo)와 결합하는 제 1 후방 어댑터, 후방 시퀀싱 프라이머와 결합하는 제 2 후방 어댑터, 및 목표 유전자의 후방 유전자와 결합하는 후방 유전자 특이적 서열을 나열된 순으로 포함하는 후방 프라이머를 포함하는 것을 특징으로 한다.The first rear adapter that binds to the rear oligo immobilized on the flow cell, the second rear adapter that binds to the rear sequencing primer, and the rear gene specific sequence that binds to the rear gene of the target gene It characterized in that it comprises a rear primer to include.

또한, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 제 2 전방 어댑터와 전방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 전방 혼합공간서열을 추가로 포함할 수 있다.In addition, the primer set for the library for sequencing of the present invention may further include a front mixed space sequence for improving sequence diversity between the second front adapter and the front gene specific sequence.

또한, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 제 2 후방 어댑터와 후방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 후방 혼합공간서열을 추가로 포함할 수 있다.In addition, the primer set for the library for sequencing of the present invention may further include a rear mixing space sequence for improving sequence diversity between the second rear adapter and the rear gene specific sequence.

또한, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 제 1 전방 어댑터와 전방 유전자 특이적 서열 사이, 구체적으로 제 1 전방 어댑터와 제 2 전방 어댑터 사이, 제 2 전방 어댑터와 전방 혼합공간서열 사이, 및 전방 혼합공간서열과 전방 유전자 특이적 서열 사이로 이루어진 군에서 선택된 위치에, 샘플의 유래 확인을 용이하게 하는 전방 표지인식서열을 추가로 포함할 수 있다.In addition, the primer set for the library for sequencing of the present invention is between the first anterior adapter and anterior gene specific sequence, specifically between the first anterior adapter and the second anterior adapter, between the second anterior adapter and anterior mixed spatial sequence And, at a position selected from the group consisting of an anterior mixed space sequence and an anterior gene specific sequence, an anterior marker recognition sequence may be further included to facilitate identification of the origin of the sample.

또한, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 제 1 후방 어댑터와 후방 유전자 특이적 서열 사이, 구체적으로 제 1 후방 어댑터와 제 2 후방 어댑터 사이, 제 2 후방 어댑터와 후방 혼합공간서열 사이, 및 후방 혼합공간서열과 후방 유전자 특이적 서열 사이로 이루어진 군에서 선택된 위치에, 샘플의 유래 확인을 용이하게 하는 후방 표지인식서열을 추가로 포함할 수 있다.In addition, the primer set for the library for sequencing of the present invention is between the first rear adapter and the rear gene specific sequence, specifically between the first rear adapter and the second rear adapter, between the second rear adapter and the rear mixed spatial sequence And, at a position selected from the group consisting of a posterior mixing space sequence and a posterior gene specific sequence, a back label recognition sequence may be further included to facilitate identification of the origin of the sample.

또한, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 전방 혼합공간서열과 전방 유전자 특이적 서열 사이에 전방 링커를 추가로 포함할 수 있다.In addition, the primer set for the library for sequencing of the present invention may further comprise a front linker between the front mixing space sequence and the front gene specific sequence.

또한, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 후방 혼합공간서열과 후방 유전자 특이적 서열 사이에 후방 링커를 추가로 포함할 수 있다.In addition, the primer set for the library for sequencing of the present invention may further include a rear linker between the rear mixing space sequence and the rear gene specific sequence.

또한, 제 1 전방 어댑터는 서열번호 1의 서열일 수 있다.In addition, the first forward adapter may be the sequence of SEQ ID NO: 1.

또한, 제 1 후방 어댑터는 서열번호 2의 서열일 수 있다.The first rear adapter may also be the sequence of SEQ ID NO: 2.

또한, 전방 표지인식서열은 서열번호 3 내지 10 중 어느 한 서열일 수 있다.In addition, the front label recognition sequence may be any one of SEQ ID NO: 3 to 10.

또한, 후방 표지인식서열은 서열번호 11 내지 22 중 어느 한 서열일 수 있다.In addition, the back label recognition sequence may be any one of SEQ ID NO: 11 to 22.

또한, 전방 혼합공간서열 또는 후방 혼합공간서열을 구성하는 염기는 In addition, the base constituting the front mixed space sequence or the rear mixed space sequence

(조건 1) 전방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 23개이고, 후방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 15임, 또는 (Condition 1) the base constituting the front mixed space sequence is absent or 1 to 23, and the base constituting the rear mixed space sequence is absent or 1 to 15, or

(조건 2) 전방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 15개이고, 후방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 23임을 만족할 수 있다.(Condition 2) The base constituting the front mixed space sequence may be absent or 1 to 15, and the base constituting the rear mixed space sequence may be absent or 1 to 23.

한편, 본 발명의 염기서열분석용 라이브러리의 제조방법은 On the other hand, the method for producing a sequencing library of the present invention

(A) 플로우셀 (flow cell)에 고정된 전방 올리고(oligo)와 결합하는 제 1 전방 어댑터, 전방 시퀀싱 프라이머와 결합하는 제 2 전방 어댑터, 및 목표 유전자의 전방 유전자와 결합하는 전방 유전자 특이적 서열을 나열된 순으로 포함하는 전방 프라이머; 및 플로우셀 (flow cell)에 고정된 후방 올리고(oligo)와 결합하는 제 1 후방 어댑터, 후방 시퀀싱 프라이머와 결합하는 제 2 후방 어댑터, 및 목표 유전자의 후방 유전자와 결합하는 후방 유전자 특이적 서열을 나열된 순으로 포함하는 후방 프라이머를 분석하고자 하는 염기서열의 양단에 결합시키는 단계, (A) a first anterior adapter that binds an anterior oligo immobilized to a flow cell, a second anterior adapter that binds an anterior sequencing primer, and an anterior gene specific sequence that binds an anterior gene of a target gene A front primer comprising in the order listed; And a first rear adapter that binds to a posterior oligo immobilized on a flow cell, a second rear adapter that binds to a rear sequencing primer, and a rear gene specific sequence that binds to the rear gene of a target gene. Binding the rear primer including the sequence to both ends of the base sequence to be analyzed;

(B) 상기 전방 프라이머 및 후방 프라이머가 양단에 결합된 염기서열을 연쇄중합효소반응으로 증폭시키는 단계, 및 (B) amplifying a nucleotide sequence in which the front and rear primers are coupled at both ends by a chain polymerase reaction, and

(C) 상기 증폭된 염기서열을 정제하는 단계를 포함하는 것을 특징으로 한다.(C) characterized in that it comprises the step of purifying the amplified base sequence.

또한, 본 발명의 염기서열분석용 라이브러리의 제조방법은 제 2 전방 어댑터와 전방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 전방 혼합공간서열을 추가로 포함할 수 있다.In addition, the method for preparing a sequencing library of the present invention may further include a front mixed space sequence for improving sequence diversity between the second front adapter and the front gene specific sequence.

또한, 본 발명의 염기서열분석용 라이브러리의 제조방법은 제 2 후방 어댑터와 후방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 후방 혼합공간서열을 추가로 포함할 수 있다.In addition, the method for preparing a sequencing library of the present invention may further include a rear mixing space sequence for improving sequence diversity between the second rear adapter and the rear gene specific sequence.

또한, 본 발명의 염기서열분석용 라이브러리의 제조방법은 제 1 전방 어댑터와 전방 유전자 특이적 서열 사이, 구체적으로 제 1 전방 어댑터와 제 2 전방 어댑터 사이, 제 2 전방 어댑터와 전방 혼합공간서열 사이, 및 전방 혼합공간서열과 전방 유전자 특이적 서열 사이로 이루어진 군에서 선택된 위치에, 샘플의 유래 확인을 용이하게 하는 전방 표지인식서열을 추가로 포함할 수 있다.In addition, the method for preparing a sequencing library of the present invention includes a first anterior adapter and an anterior gene specific sequence, specifically, between a first anterior adapter and a second anterior adapter, between a second anterior adapter and an anterior mixed spatial sequence, And at a position selected from the group consisting of an anterior mixed space sequence and an anterior gene specific sequence, an anterior label recognition sequence for facilitating identification of a sample.

또한, 본 발명의 염기서열분석용 라이브러리의 제조방법은 제 1 후방 어댑터와 후방 유전자 특이적 서열 사이, 구체적으로 제 1 후방 어댑터와 제 2 후방 어댑터 사이, 제 2 후방 어댑터와 후방 혼합공간서열 사이, 및 후방 혼합공간서열과 후방 유전자 특이적 서열 사이로 이루어진 군에서 선택된 위치에, 샘플의 유래 확인을 용이하게 하는 후방 표지인식서열을 추가로 포함할 수 있다.In addition, the method for preparing a sequencing library of the present invention is provided between a first rear adapter and a rear gene specific sequence, specifically, between a first rear adapter and a second rear adapter, between a second rear adapter and a rear mixed spatial sequence, And at a position selected from the group consisting of a posterior mixing space sequence and a posterior gene specific sequence, a rear labeling sequence that facilitates identification of the origin of the sample.

또한, 본 발명의 염기서열분석용 라이브러리의 제조방법은 상기 증폭시키는 단계 및 정제하는 단계를 각각 1 회씩만 포함할 수 있다.In addition, the method for preparing a sequencing library of the present invention may include only one amplification step and one purification step.

본 발명의 시퀀싱 라이브러리 제조방법을 이용할 경우 종래 두 번 이상 수행해야 했던 연쇄중합효소반응과 핵산정제과정을 각각 한 번씩만 수행하는 것이 가능해졌다. 그 결과 주형을 증폭하는 시간까지 포함하여 약 2 시간 내외로 라이브러리를 제조할 수 있게 되었다.When the sequencing library manufacturing method of the present invention is used, it is possible to perform the chain polymerase reaction and the nucleic acid purification process that had to be performed two or more times only once. As a result, the library can be prepared in about 2 hours including the time for amplifying the template.

또한, 본 발명으로 제조한 시퀀싱 라이브러리는 일루미나사의 차세대 염기서열 분석기 뿐만 아니라 그와 동일한 원리를 이용하는 분석기에도 사용할 수 있다. In addition, the sequencing library prepared by the present invention can be used not only for the next-generation sequencing analyzer of Illumina Corporation, but also for analyzers using the same principle.

또한 공정을 최소화하여 염기서열 분석결과의 왜곡 발생 가능성을 최소화하였으며 기존 제조방법보다 간단하게 시퀀싱 라이브러리를 제조하는 장점이 있다.In addition, by minimizing the process, the possibility of distortion of the sequencing results is minimized, and the sequencing library can be manufactured more simply than the conventional method.

도 1은 종래 프라이머를 활용한 시퀀싱 라이브러리의 제조공정을 나타내는 개념도이다.1 is a conceptual diagram illustrating a manufacturing process of a sequencing library using a conventional primer.

도 2는 본 발명의 융합 프라이머를 활용한 시퀀싱 라이브러리의 제조공정의 일 실시예를 나타내는 개념도이다.Figure 2 is a conceptual diagram showing an embodiment of the manufacturing process of the sequencing library using the fusion primer of the present invention.

이하, 본 발명의 바람직한 실시예에 대하여 상세히 설명한다. 또한, 하기의 설명에서는 구체적인 구성요소 등과 같은 많은 특정사항들이 설명되어 있는데, 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐 이러한 특정 사항들 없이도 본 발명이 실시될 수 있음은 이 기술분야에서 통상의 지식을 가진 자에게는 자명하다 할 것이다. 그리고, 본 발명을 설명함에 있어서, 관련된 공지 기능 혹은 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, many specific details such as specific components are described in the following description, which is provided to help a more general understanding of the present invention, and the present invention may be practiced without these specific details. It is self-evident to those who have knowledge of the world. In describing the present invention, when it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

본 발명에서는 상술한 바와 같은 목적을 달성하기 위하여, 융합 프라이머를 이용하여 한번의 연쇄중합효소반응과 한번의 정제과정을 거쳐 시퀀싱 라이브러리를 제조한다. 융합 프라이머는 전방 프라이머와 후방 프라이머로 이루어져 있으며, 각각의 프라이머는 다시 플로우셀 (flow cell)에 고정된 올리고(oligo)와 결합하는 제 1 어댑터 (170, 270), 샘플의 유래 확인을 용이하게 하는 표지인식서열 (160, 260), 시퀀싱 프라이머와 결합하는 제 2 어댑터 (150, 250), 서열 다양성 향상을 위한 혼합공간서열 (140, 240), 주형 (10)과 결합하는 유전자 특이 염기서열 (120, 220), 유전자 특이 염기서열 (120, 220)을 나머지 서열과 연결하는 링커 (130, 230)를 포함한다. 표지 인식이 필요하지 않을 경우 표지인식서열 (160, 260)은 제외될 수 있으며 자체 제작 시퀀싱 프라이머를 사용할 경우 제 2 어댑터 (150, 250)는 변경될 수 있다. 링커 (130, 230)는 목표 유전자 특이 염기서열과 그외의 부분을 연결시켜 주는 염기서열로써 염기서열의 구성이나 길이는 다양하게 구성할 수 있다.In the present invention, to achieve the object as described above, using a fusion primer to prepare a sequencing library through one chain polymerase reaction and one purification process. The fusion primer consists of a front primer and a back primer, each primer again to the first adapter (170, 270) that binds to the oligo immobilized in the flow cell, to facilitate the origin of the sample Label recognition sequences (160, 260), second adapters (150, 250) to bind to sequencing primers, mixed spatial sequences (140, 240) to enhance sequence diversity, gene specific sequences (120) to bind to template (10) , 220), linker (130, 230) for connecting the gene-specific nucleotide sequence (120, 220) with the rest of the sequence. If label recognition is not required, the label recognition sequences 160 and 260 may be excluded, and the second adapters 150 and 250 may be changed using a self-made sequencing primer. The linkers 130 and 230 are nucleotide sequences connecting the target gene specific nucleotide sequences and other portions thereof, and may have various configurations or lengths of the nucleotide sequences.

구체적으로, 본 발명의 염기서열분석을 위한 라이브러리용 프라이머 세트는 전방 프라이머 및 후방 프라이머를 포함하는데, 각각의 프라이머는 제 1 어댑터 (170, 270), 제 2 어댑터 (150, 250), 및 유전자 특이적 서열 (120, 220)을 나열된 순으로 포함한다.Specifically, the primer set for the library for sequencing of the present invention includes a front primer and a back primer, each primer is a first adapter (170, 270), a second adapter (150, 250), and gene specific The enemy sequences 120, 220 are included in the order listed.

그리고, 상기 각각의 프라이머는 제 2 어댑터 (150, 250)와 유전자 특이적 서열 (120, 220) 사이에 혼합공간서열 (140, 240)을, 그리고 제 1 어댑터 (170, 270)와 유전자 특이적 서열 (120, 220) 사이에 표지인식서열 (160, 260)을, 또한 유전자 특이적 서열 (120, 220)과 나머지 서열 사이에 링커 (130, 230)를 추가로 포함할 수 있다.In addition, each of the primers has a mixed spatial sequence (140, 240) between the second adapter (150, 250) and the gene specific sequence (120, 220), and the first adapter (170, 270) and gene specific Labeling sequences 160 and 260 between the sequences 120 and 220 and also linkers 130 and 230 between the gene specific sequences 120 and 220 and the remaining sequences.

이처럼 하나의 프라이머에 상이한 역할을 가진 서열들을 서로 모아서 융합시킨 다음 이를 주형 (10) 양 말단에 결합시킴으로써 연쇄중합효소반응 및 정제과정의 반복 횟수를 줄이는 것이 본 발명의 가장 큰 특징이다. 이러한 반복 횟수의 저감은 우선 시퀀싱 라이브러리 제작에 소요되는 시간을 단축시킬 뿐만 아니라 시료의 왜곡이나 손실 등을 방지하고 비숙련자도 능숙하게 라이브러리를 제작할 수 있게 한다.As such, the greatest feature of the present invention is to reduce the number of repetitions of the chain polymerase reaction and purification process by collecting and fusing sequences having different roles in one primer and then binding them to both ends of the template (10). This reduction in the number of iterations not only shortens the time required to produce the sequencing library, but also prevents distortion or loss of the sample and enables the skilled person to produce the library skillfully.

본 발명의 융합 프라이머를 구성하는 서열들 중 제 1 어댑터 (170, 270)는 증폭을 위한 플로우셀 (flow cell)에 고정된 올리고(oligo), 즉 올리고뉴클레오티드와 결합함으로써 전술한 브릿지 연쇄중합효소반응을 가능하게 한다. 본 발명은 이러한 제 1 어댑터 (170, 270)로서 주형 (10)의 전방에 위치하는 제 1 전방 어댑터 (170)와 주형 (10)의 후방에 위치하는 제 1 후방 어댑터 (270)를 포함한다. 그리고, 본 발명의 제 1 전방 어댑터 (170)는 서열번호 1의 서열일 수 있고, 제 1 후방 어댑터 (270)는 서열번호 2의 서열일 수 있다.Among the sequences constituting the fusion primer of the present invention, the first adapters 170 and 270 bind to oligos, i.e., oligonucleotides, immobilized in a flow cell for amplification. To make it possible. The present invention includes, as such first adapters 170 and 270, a first front adapter 170 located in front of the mold 10 and a first rear adapter 270 located behind the mold 10. And, the first front adapter 170 of the present invention may be a sequence of SEQ ID NO: 1, the first rear adapter 270 may be a sequence of SEQ ID NO: 2.

제 2 어댑터 (150, 250)는 염기서열 분석을 위한 시퀀싱 프라이머와 결합하는 역할을 수행하며, 따라서 사용하는 시퀀싱 프라이머에 맞게 상보적인 서열을 갖도록 변경될 수 있다. 본 발명의 제 2 전방 어댑터 (150)는 전방 시퀀싱 프라이머와 결합하고, 제 2 후방 어댑터 (250)는 후방 시퀀싱 프라이머와 결합한다.The second adapter 150, 250 serves to bind the sequencing primer for sequencing, and thus may be modified to have a complementary sequence for the sequencing primer used. The second front adapter 150 of the present invention binds to the front sequencing primer, and the second back adapter 250 binds to the back sequencing primer.

그리고, 유전자 특이적 서열 (120, 220)은 주형 (10)의 양 말단 유전자와 결합할 수 있도록 하는 것으로서 주형 (10)에 따라 그 서열이 변경될 수 있다.In addition, the gene specific sequences 120 and 220 may bind to both terminal genes of the template 10, and the sequence may be changed according to the template 10.

본 발명은 이들 제 1 어댑터 (170, 270), 제 2 어댑터 (150, 250) 및 유전자 특이적 서열 (120, 220)이 서로 융합되어 하나의 프라이머에 존재함으로써, 염기서열을 중간중간 끊어서 분석하지 않고 죽 이어서 한번에 분석하는 것이 가능하다.According to the present invention, since the first adapter (170, 270), the second adapter (150, 250) and the gene specific sequence (120, 220) are fused to each other and exist in one primer, the nucleotide sequence is not interrupted. Without killing it is possible to analyze at once.

본 발명은 또한 상기 제 2 어댑터 (150, 250)와 유전자 특이적 서열 (120, 220) 사이에 혼합공간서열 (140, 240)을 추가로 구비할 수 있다. 이 혼합공간서열 (140, 240)은 분석하고자 하는 서열들이 서로 유사하여 다양성(diversity)이 낮은 경우 분석의 정확도가 급감하는 것을 방지하는 역할을 한다. The present invention may further include a mixed spatial sequence (140, 240) between the second adapter (150, 250) and the gene specific sequence (120, 220). The mixed spatial sequences 140 and 240 serve to prevent a sudden drop in the accuracy of analysis when the sequences to be analyzed are similar to each other and thus have low diversity.

상기 전방 혼합공간서열 (140) 또는 후방 혼합공간서열 (240)을 구성하는 염기는 (조건 1) 전방 혼합공간서열 (140)을 구성하는 염기는 부재하거나 1 내지 23개이고, 후방 혼합공간서열 (240)을 구성하는 염기는 부재하거나 1 내지 15임, 또는 (조건 2) 전방 혼합공간서열 (140)을 구성하는 염기는 부재하거나 1 내지 15개이고, 후방 혼합공간서열 (240)을 구성하는 염기는 부재하거나 1 내지 23임을 만족하는 것을 특징으로 한다. 이러한 혼합공간서열 (140, 240)의 존재로 인해 전술한 미생물 군집체와 같이 서열 간 다양성이 낮은 경우 분석의 정확도를 현저히 상승시킬 수 있다. 본 발명에서는 제 2 전방 어댑터 (150)와 전방 유전자 특이적 서열 (120) 사이에 전방 혼합공간서열 (140)을, 제 2 후방 어댑터 (250)와 후방 유전자 특이적 서열 (220) 사이에 후방 혼합공간서열 (240)을 추가로 포함할 수 있다.The base constituting the front mixed space sequence 140 or the rear mixed space sequence 240 is (condition 1) the base constituting the front mixed space sequence 140 is absent or 1 to 23, and the rear mixed space sequence 240 ) Base is absent or is 1 to 15, or (condition 2) bases constituting the front mixed space sequence 140 is absent or 1 to 15 bases, and bases constituting the rear mixed space sequence 240 is absent Or to satisfy 1 to 23. Due to the presence of such mixed spatial sequences (140, 240) it is possible to significantly increase the accuracy of the analysis when the diversity between the sequences, such as the microbial population described above is low. In the present invention, the rear mixed space sequence 140 is mixed between the second anterior adapter 150 and the anterior gene specific sequence 120, and the rear mixed between the second rear adapter 250 and the rear gene specific sequence 220. The spatial sequence 240 may further include.

본 발명은 제 1 어댑터 (170, 270)와 유전자 특이적 서열 (120, 220) 사이에 표지인식서열 (160, 260)을 추가로 구비할 수 있는데, 상기 표지인식서열 (160, 260)은 분석되고 있는 서열이 어느 샘플에서 유래하였는지 확인을 용이하게 하는 역할을 한다. 본 발명에서는 제 1 전방 어댑터 (170)와 전방 유전자 특이적 서열 (120) 사이, 구체적으로 제 1 전방 어댑터 (170)와 제 2 전방 어댑터 (150) 사이, 제 2 전방 어댑터 (150)와 전방 혼합공간서열 (140) 사이, 및 전방 혼합공간서열 (140)과 전방 유전자 특이적 서열 (120) 사이로 이루어진 군에서 선택된 위치에 전방 표지인식서열 (160)을 추가로 포함할 수 있고, 제 1 후방 어댑터 (270)와 후방 유전자 특이적 서열 (220) 사이, 구체적으로 제 1 후방 어댑터 (270)와 제 2 후방 어댑터 (250) 사이, 제 2 후방 어댑터 (250)와 후방 혼합공간서열 (240) 사이, 및 후방 혼합공간서열 (240)과 후방 유전자 특이적 서열 (220) 사이로 이루어진 군에서 선택된 위치에 후방 표지인식서열 (260)을 추가로 포함할 수 있다. 본 발명에서 상기 전방 표지인식서열 (160)은 서열번호 3 내지 10 중 어느 한 서열일 수 있고, 후방 표지인식서열 (260)은 서열번호 11 내지 22 중 어느 한 서열일 수 있다.The present invention may further include a label recognition sequence (160, 260) between the first adapter (170, 270) and the gene specific sequence (120, 220), the label recognition sequence (160, 260) is analyzed It serves to facilitate identification of which sample the sequence is being derived from. In the present invention, forward mixing between the first anterior adapter 170 and the anterior gene specific sequence 120, specifically, between the first anterior adapter 170 and the second anterior adapter 150, is forward mixed with the second anterior adapter 150. And may further comprise an anterior marker sequence 160 at a position selected between the spatial sequences 140 and between the anterior mixed spatial sequence 140 and the anterior gene specific sequence 120, the first posterior adapter. Between 270 and posterior gene specific sequence 220, specifically between first posterior adapter 270 and second posterior adapter 250, between second posterior adapter 250 and posterior mixed spatial sequence 240, And a rear label recognition sequence 260 at a position selected from the group consisting of a rear mixing space sequence 240 and a rear gene specific sequence 220. In the present invention, the front label recognition sequence 160 may be any one of SEQ ID NOs: 3 to 10, and the rear label identification sequence 260 may be any one of SEQ ID NOs: 11 to 22.

본 발명은 나아가 유전자 특이적 서열 (120, 220)을 링커 (130, 230)를 통해 나머지 서열들과 연결시킬 수도 있다. 본 발명에서는 전방 혼합공간서열 (140)과 전방 유전자 특이적 서열 (120) 사이에 전방 링커 (130)를, 후방 혼합공간서열 (240)과 후방 유전자 특이적 서열 (220) 사이에 후방 링커 (230)를 추가로 포함할 수 있다.The invention may further link gene specific sequences 120 and 220 with the remaining sequences via linkers 130 and 230. In the present invention, the front linker 130 between the front mixed space sequence 140 and the front gene specific sequence 120, and the rear linker 230 between the rear mixed space sequence 240 and the rear gene specific sequence 220. ) May be further included.

한편, 본 발명의 염기서열분석용 라이브러리의 제조방법은 On the other hand, the method for producing a sequencing library of the present invention

(A) 전술한 전방 프라이머와 후방 프라이머를 분석하고자 하는 염기서열 즉 주형 (10)의 양단에 결합시키는 단계, (A) binding the aforementioned front primer and the rear primer to both ends of the base sequence to be analyzed, that is, the template (10),

(B) 상기 전방 프라이머 및 후방 프라이머가 양단에 결합된 염기서열을 연쇄중합효소반응으로 증폭시키는 단계, 및 (B) amplifying a nucleotide sequence in which the front and rear primers are coupled at both ends by a chain polymerase reaction, and

(C) 상기 증폭된 염기서열을 정제하는 단계를 포함하는 것을 특징으로 한다.(C) characterized in that it comprises the step of purifying the amplified base sequence.

특히, 본 발명의 염기서열분석용 라이브러리의 제조방법은 종래기술들과 달리 상기 증폭시키는 단계 및 정제하는 단계를 각각 1 회씩만 포함할 수 있다. 즉, 본 발명은 전술한 융합 프라이머를 사용함으로써 염기서열 분석을 중간중간 끊어서 수회 반복하지 않고 한번에 죽 이어서 1 회에 완료하는 것이 가능하다.In particular, the method for preparing a sequencing library of the present invention may include only the amplifying and purifying steps only once, unlike the prior arts. That is, according to the present invention, the sequencing primer can be used to kill sequencing at once and complete at once without interrupting sequencing.

이하에서 설명되는 실시예는 미생물 군집 유전체에서 16S rRNA 유전자 기반 군집 분석을 통하여 군집 조성비를 구하였다. Example described below was to determine the population composition ratio through 16S rRNA gene-based cluster analysis in the microbial community genome.

실시예Example

제조예 : 16S rRNA 유전자의 확보 및 인공군집체(mock community)구성Preparation Example: Securing 16S rRNA gene and constructing mock community

채취한 환경시료에서 토양미생물유전체 추출키트(MO BIO, 미국)를 사용하여 시료내의 미생물 염색체를 추출하였다. 추출된 염색체를 주형(template)로 하여 연쇄효소중합반응(PCR)을 통해 16S rRNA 유전자를 증폭하였다. 16S rRNA 특이적 프라이머로 다양한 분류군에서 공통적으로 쓰이는 bac27f(AGAGTTTGATCMTGGCTCAG), bac1492r(GGTTACCTTGTTACGACTT)를 사용하여 16S rRNA 유전자를 증폭했다. 증폭된 유전자는 pGEM-T easy 벡터(Promega, 미국)에 접합시켜 대장균을 형질전환하여 클로닝을 수행했다. 플라스미드 DNA는 클로닝을 통해 확보한 재조합 대장균에서 Qiagen plasmid mini kit (Qiagen, 독일)를 활용하여 추출하였다. 확보된 플라스미드는 pGEM-T easy 벡터 특이적 프라이머를 이용, Sybr Green qPCR을 통하여 정량하였다. The microbial chromosome in the sample was extracted from the sample collected using soil microbial dielectric kit (MO BIO, USA). Using the extracted chromosome as a template, 16S rRNA gene was amplified by PCR. 16S rRNA genes were amplified using bac27f (AGAGTTTGATCMTGGCTCAG) and bac1492r (GGTTACCTTGTTACGACTT) commonly used in various taxa as 16S rRNA specific primers. The amplified gene was conjugated to the pGEM-T easy vector (Promega, USA) to transform E. coli to perform cloning. Plasmid DNA was extracted from the recombinant E. coli obtained by cloning using the Qiagen plasmid mini kit (Qiagen, Germany). The obtained plasmid was quantified by Sybr Green qPCR using pGEM-T easy vector specific primers.

실시예 : 융합 프라이머를 이용한 16S rRNA 유전자의 증폭 및 시퀀싱 라이브러리의 준비Example: Preparation of 16S rRNA Gene Amplification and Sequencing Library Using Fusion Primers

상기 제조예에서 확보된 16S rRNA 유전자를 주형으로 하여 융합 프라이머로 연쇄중합반응을 진행, 연쇄중합반응 조건은 표 1과 같다. 융합 프라이머의 서열정보는 표 2, 표 3, 표 4와 같다. 기존 프라이머를 이용한 시퀀싱 라이브러리 제조공정과 차이점은 도 1 및 도 2로부터 확인할 수 있다.Using the 16S rRNA gene secured in the preparation example as a template, the chain polymerization proceeds with a fusion primer, the chain polymerization reaction conditions are shown in Table 1. Sequence information of the fusion primers are shown in Table 2, Table 3, Table 4. Differences from the sequencing library manufacturing process using conventional primers can be seen from FIGS. 1 and 2.

표 1 PCR 반응조건 PCR 반응조건 초기 변성 94℃ 5분 1회 변성 94℃ 30초 25회 어닐링 55℃ 30초 연장 72℃ 90초 최종 연장 72℃ 5분 1회 Table 1 PCR reaction condition PCR reaction condition Early degeneration 94 ℃ 5 minutes 1 time denaturalization 94 ℃ 30 seconds 25 times Annealing 55 ℃ 30 seconds extension 72 ℃ 90 sec Final extension 72 ℃ 5 minutes 1 time

확보된 증폭산물(amplicon)은 AMPure XP beads (Agencourt, 프랑스)를 사용하여 정제한 후 qPCR로 정량하고 각각의 정제산물을 pooling하여 시퀀싱 라이브러리로 사용하였다.The amplified product (amplicon) was purified using AMPure XP beads (Agencourt, France), quantified by qPCR and pooled each purified product was used as a sequencing library.

표 2 전방 프라이머 (Bac27f) 제 1 전방 어댑터 전방 표지인식서열 제 2 전방 어댑터 전방 혼합공간서열 전방링커 AATGATACGGCGACCACCGAGATCTACAC TAGATCGC TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG AC AATGATACGGCGACCACCGAGATCTACAC CTCTCTAT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG C AC AATGATACGGCGACCACCGAGATCTACAC TATCCTCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GT AC AATGATACGGCGACCACCGAGATCTACAC AGAGTAGA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TCG AC AATGATACGGCGACCACCGAGATCTACAC GTAAGGAG TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TGTC AC AATGATACGGCGACCACCGAGATCTACAC ACTGCATA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG ATCTT AC AATGATACGGCGACCACCGAGATCTACAC AAGGAGTA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GATGTC AC AATGATACGGCGACCACCGAGATCTACAC CTAAGCCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CGATCTC AC TABLE 2 Forward Primer (Bac27f) First front adapter Front cover recognition sequence 2nd front adapter Front mixed space sequence Front linker AATGATACGGCGACCACCGAGATCTACAC TAGATCGC TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG AC AATGATACGGCGACCACCGAGATCTACAC CTCTCTAT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG C AC AATGATACGGCGACCACCGAGATCTACAC TATCCTCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GT AC AATGATACGGCGACCACCGAGATCTACAC AGAGTAGA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TCG AC AATGATACGGCGACCACCGAGATCTACAC GTAAGGAG TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TGTC AC AATGATACGGCGACCACCGAGATCTACAC ACTGCATA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG ATCTT AC AATGATACGGCGACCACCGAGATCTACAC AAGGAGTA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GATGTC AC AATGATACGGCGACCACCGAGATCTACAC CTAAGCCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CGATCTC AC

표 3 전방 프라이머 (Bfi27f) 제 1 전방 어댑터 전방 표지인식서열 제 2 전방 어댑터 전방 혼합공간서열 전방링커 AATGATACGGCGACCACCGAGATCTACAC TAGATCGC TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG AC AATGATACGGCGACCACCGAGATCTACAC CTCTCTAT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG C AC AATGATACGGCGACCACCGAGATCTACAC TATCCTCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GT AC AATGATACGGCGACCACCGAGATCTACAC AGAGTAGA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TCG AC AATGATACGGCGACCACCGAGATCTACAC GTAAGGAG TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TGTC AC AATGATACGGCGACCACCGAGATCTACAC ACTGCATA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG ATCTT AC AATGATACGGCGACCACCGAGATCTACAC AAGGAGTA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GATGTC AC AATGATACGGCGACCACCGAGATCTACAC CTAAGCCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CGATCTC AC TABLE 3 Anterior Primer (Bfi27f) First front adapter Front cover recognition sequence 2nd front adapter Front mixed space sequence Front linker AATGATACGGCGACCACCGAGATCTACAC TAGATCGC TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG AC AATGATACGGCGACCACCGAGATCTACAC CTCTCTAT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG C AC AATGATACGGCGACCACCGAGATCTACAC TATCCTCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GT AC AATGATACGGCGACCACCGAGATCTACAC AGAGTAGA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TCG AC AATGATACGGCGACCACCGAGATCTACAC GTAAGGAG TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TGTC AC AATGATACGGCGACCACCGAGATCTACAC ACTGCATA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG ATCTT AC AATGATACGGCGACCACCGAGATCTACAC AAGGAGTA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GATGTC AC AATGATACGGCGACCACCGAGATCTACAC CTAAGCCT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CGATCTC AC

표 4 후방 프라이머 (Bac516r) 제 1 후방 어댑터 후방 표지인식서열 제 2 후방 어댑터 후방 혼합공간서열 후방링커 CAAGCAGAAGACGGCATACGAGAT TCGCCTTA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG AC CAAGCAGAAGACGGCATACGAGAT CTAGTACG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG G AC CAAGCAGAAGACGGCATACGAGAT TTCTGCCT GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG CG AC CAAGCAGAAGACGGCATACGAGAT GCTCAGGA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG TCG AC CAAGCAGAAGACGGCATACGAGAT AGGAGTCC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG ATCG AC CAAGCAGAAGACGGCATACGAGAT CATGCCTA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GATCG AC CAAGCAGAAGACGGCATACGAGAT GTAGAGAG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG CGATCG AC CAAGCAGAAGACGGCATACGAGAT CCTCTCTG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG TCGAGCG AC CAAGCAGAAGACGGCATACGAGAT AGCGTAGC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GTCGATCG AC CAAGCAGAAGACGGCATACGAGAT CAGCCTCG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG AGTCGATCG AC CAAGCAGAAGACGGCATACGAGAT TGCCTCTT GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG TAGTCGATCG AC CAAGCAGAAGACGGCATACGAGAT TCCTCTAC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG CTAGTCGATTG AC Table 4 Rear Primer (Bac516r) First rear adapter Rear cover recognition sequence 2nd rear adapter Rear mixed space sequence Rear linker CAAGCAGAAGACGGCATACGAGAT TCGCCTTA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG AC CAAGCAGAAGACGGCATACGAGAT CTAGTACG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG G AC CAAGCAGAAGACGGCATACGAGAT TTCTGCCT GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG CG AC CAAGCAGAAGACGGCATACGAGAT GCTCAGGA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG TCG AC CAAGCAGAAGACGGCATACGAGAT AGGAGTCC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG ATCG AC CAAGCAGAAGACGGCATACGAGAT CATGCCTA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GATCG AC CAAGCAGAAGACGGCATACGAGAT GTAGAGAG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG CGATCG AC CAAGCAGAAGACGGCATACGAGAT CCTCTCTG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG TCGAGCG AC CAAGCAGAAGACGGCATACGAGAT AGCGTAGC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GTCGATCG AC CAAGCAGAAGACGGCATACGAGAT CAGCCTCG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG AGTCGATCG AC CAAGCAGAAGACGGCATACGAGAT TGCCTCTT GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG TAGTCGATCG AC CAAGCAGAAGACGGCATACGAGAT TCCTCTAC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG CTAGTCGATTG AC

시험예 : 차세대 염기서열분석을 통한 염기서열 확보 및 분석Test Example: Securing and analyzing nucleotide sequences through next-generation sequencing

상기 실시예에서 확보된 시퀀싱 라이브러리는 마이씩 (Illumina, 미국)에 300bp PE (v3) 시퀀싱 키트를 사용하여 염기서열정보를 확보하였다. In the sequencing library obtained in the above example, the nucleotide sequence information was obtained using a 300bp PE (v3) sequencing kit in Mythal (Illumina, USA).

확보된 염기서열은 PANDAseq, MOTHUR를 통하여 분류군별로 분석되었으며 그 결과는 표 5와 같다.The obtained nucleotide sequences were analyzed by classification groups through PANDAseq and MOTHUR, and the results are shown in Table 5.

표 5 혼합공간서열을 설정한 융합 프라이머로 증폭한 결과 Taxon Mock6(%) Mock7(%) Mock8(%) Mock9(%) Mock10(%) Burkholderia_phytofirmans 2.14±0.40 0.02±0.00 3.59±0.81 0.01±0.01 0.05±0.02 EU150278_s 1.60±0.48 0.02±0.01 2.64±0.48 0.01±0.01 0.03±0.01 EU335310_s 6.37±1.00 0.14±0.04 7.79±1.39 0.05±0.02 0.07±0.04 Mycobacterium_fluoranthenivorans 3.64±1.00 4.49±0.53 0.06±0.03 0.09±0.05 0.05±0.03 DQ136121_s 1.22±0.23 0.00±0.00 0.01±0.00 0.55±0.03 0.01±0.01 EF520485_s 1.79±0.55 0.03±0.01 2.09±0.40 0.02±0.01 0.06±0.02 FJ889215_s 0.66±0.12 0.77±0.06 0.02±0.02 0.78±0.07 0.01±0.01 EU335343_s 2.93±1.07 0.12±0.05 4.68±1.29 0.04±0.02 6.84±0.56 HM748719_s 2.06±0.18 4.86±0.45 0.16±0.09 4.39±0.60 0.02±0.01 Paenibacillus_pabuli 0.45±0.04 1.09±0.12 1.00±0.07 0.01±0.01 0.01±0.00 Merismopedia 0.01±0.01 0.00±0.00 0.00±0.00 0.05±0.02 0.02±0.00 DQ404777_s 0.37±0.10 0.01±0.01 0.88±0.20 0.01±0.01 0.01±0.00 AJ617867_s 1.45±0.39 0.04±0.02 0.09±0.04 2.45±0.65 0.05±0.02 EU979093_s 4.74±0.60 0.07±0.02 0.13±0.07 12.28±1.89 11.11±0.79 EU335174_s 0.78±0.19 1.97±0.85 0.05±0.03 1.61±0.37 0.01±0.00 EU331380_s 2.30±0.10 4.32±0.81 0.21±0.15 5.51±1.14 5.92±0.45 DQ404764_s 0.98±0.47 0.05±0.02 0.08±0.04 2.30±1.11 2.68±1.68 AY921508_s 5.66±2.41 0.13±0.04 0.14±0.05 14.06±3.72 0.25±0.07 AM991247_s 2.51±0.64 5.55±0.95 0.11±0.07 0.08±0.03 5.04±0.81 JN023782_s 1.95±0.42 3.92±0.87 0.04±0.02 0.02±0.01 4.90±0.77 EF492921_s 5.89±1.11 11.96±2.06 12.37±1.82 11.95±1.17 0.31±0.12 EU305673_s 2.60±0.34 4.05±0.46 2.57±0.24 0.04±0.03 3.70±0.54 EU386125_s 0.88±0.09 0.01±0.00 1.54±0.16 0.01±0.01 0.01±0.01 Methylocystis_rosea 0.19±0.07 0.01±0.01 0.21±0.10 0.00±0.00 0.00±0.00 DQ404639_s 0.49±0.21 0.84±0.29 0.03±0.01 1.07±0.33 0.59±0.27 EF516883_s 4.76±0.79 10.86±0.99 10.28±0.69 13.14±0.19 0.07±0.03 4P002353_s 3.96±1.80 5.80±1.36 0.17±0.13 0.03±0.02 0.07±0.04 AB240240_s 2.08±0.27 0.03±0.02 3.45±0.58 0.02±0.01 4.33±0.52 AY921992_s 1.65±0.30 0.02±0.01 2.67±0.53 0.02±0.02 0.02±0.01 AM696938_s 5.64±0.85 0.08±0.04 9.40±1.49 0.05±0.03 15.67±2.19 FJ810576_s 2.41±0.82 0.05±0.03 3.51±0.82 0.01±0.01 5.80±1.00 AB672115_s 4.57±0.29 6.62±0.47 4.13±0.36 7.18±0.66 5.69±0.33 EF492955_s 0.80±0.13 1.59±0.19 0.05±0.03 1.37±0.19 1.73±0.27 Clostridium_saccharoperbutylacetonicum 5.34±0.32 9.98±0.82 9.62±0.84 0.13±0.06 0.13±0.05 AB240328_s 1.65±0.36 3.78±0.57 3.53±0.48 3.82±0.42 3.85±0.90 FJ936969_s 0.91±0.26 0.01±0.01 1.43±0.41 0.02±0.01 0.01±0.00 AY221607_s 1.23±0.30 2.45±0.78 2.01±0.65 2.33±0.63 0.03±0.01 FJ793157_s 6.24±0.54 8.53±0.38 8.01±0.28 9.64±1.37 15.72±0.92 AB672259_s 0.96±0.19 2.35±0.16 0.02±0.02 0.01±0.00 0.02±0.01 AY921885_s 2.03±0.48 0.10±0.05 0.07±0.02 4.41±1.25 4.53±0.90 unclassified 2.11±0.67 3.31±0.55 1.20±0.77 0.44±0.25 0.60±0.28 Table 5 Result of amplification with fusion primer that set mixed spatial sequence Taxon Mock6 (%) Mock7 (%) Mock8 (%) Mock9 (%) Mock10 (%) Burkholderia_phytofirmans 2.14 ± 0.40 0.02 ± 0.00 3.59 ± 0.81 0.01 ± 0.01 0.05 ± 0.02 EU150278_s 1.60 ± 0.48 0.02 ± 0.01 2.64 ± 0.48 0.01 ± 0.01 0.03 ± 0.01 EU335310_s 6.37 ± 1.00 0.14 ± 0.04 7.79 ± 1.39 0.05 ± 0.02 0.07 ± 0.04 Mycobacterium_fluoranthenivorans 3.64 ± 1.00 4.49 ± 0.53 0.06 ± 0.03 0.09 ± 0.05 0.05 ± 0.03 DQ136121_s 1.22 ± 0.23 0.00 ± 0.00 0.01 ± 0.00 0.55 ± 0.03 0.01 ± 0.01 EF520485_s 1.79 ± 0.55 0.03 ± 0.01 2.09 ± 0.40 0.02 ± 0.01 0.06 ± 0.02 FJ889215_s 0.66 ± 0.12 0.77 ± 0.06 0.02 ± 0.02 0.78 ± 0.07 0.01 ± 0.01 EU335343_s 2.93 ± 1.07 0.12 ± 0.05 4.68 ± 1.29 0.04 ± 0.02 6.84 ± 0.56 HM748719_s 2.06 ± 0.18 4.86 ± 0.45 0.16 ± 0.09 4.39 ± 0.60 0.02 ± 0.01 Paenibacillus_pabuli 0.45 ± 0.04 1.09 ± 0.12 1.00 ± 0.07 0.01 ± 0.01 0.01 ± 0.00 Merismopedia 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.05 ± 0.02 0.02 ± 0.00 DQ404777_s 0.37 ± 0.10 0.01 ± 0.01 0.88 ± 0.20 0.01 ± 0.01 0.01 ± 0.00 AJ617867_s 1.45 ± 0.39 0.04 ± 0.02 0.09 ± 0.04 2.45 ± 0.65 0.05 ± 0.02 EU979093_s 4.74 ± 0.60 0.07 ± 0.02 0.13 ± 0.07 12.28 ± 1.89 11.11 ± 0.79 EU335174_s 0.78 ± 0.19 1.97 ± 0.85 0.05 ± 0.03 1.61 ± 0.37 0.01 ± 0.00 EU331380_s 2.30 ± 0.10 4.32 ± 0.81 0.21 ± 0.15 5.51 ± 1.14 5.92 ± 0.45 DQ404764_s 0.98 ± 0.47 0.05 ± 0.02 0.08 ± 0.04 2.30 ± 1.11 2.68 ± 1.68 AY921508_s 5.66 ± 2.41 0.13 ± 0.04 0.14 ± 0.05 14.06 ± 3.72 0.25 ± 0.07 AM991247_s 2.51 ± 0.64 5.55 ± 0.95 0.11 ± 0.07 0.08 ± 0.03 5.04 ± 0.81 JN023782_s 1.95 ± 0.42 3.92 ± 0.87 0.04 ± 0.02 0.02 ± 0.01 4.90 ± 0.77 EF492921_s 5.89 ± 1.11 11.96 ± 2.06 12.37 ± 1.82 11.95 ± 1.17 0.31 ± 0.12 EU305673_s 2.60 ± 0.34 4.05 ± 0.46 2.57 ± 0.24 0.04 ± 0.03 3.70 ± 0.54 EU386125_s 0.88 ± 0.09 0.01 ± 0.00 1.54 ± 0.16 0.01 ± 0.01 0.01 ± 0.01 Methylocystis_rosea 0.19 ± 0.07 0.01 ± 0.01 0.21 ± 0.10 0.00 ± 0.00 0.00 ± 0.00 DQ404639_s 0.49 ± 0.21 0.84 ± 0.29 0.03 ± 0.01 1.07 ± 0.33 0.59 ± 0.27 EF516883_s 4.76 ± 0.79 10.86 ± 0.99 10.28 ± 0.69 13.14 ± 0.19 0.07 ± 0.03 4P002353_s 3.96 ± 1.80 5.80 ± 1.36 0.17 ± 0.13 0.03 ± 0.02 0.07 ± 0.04 AB240240_s 2.08 ± 0.27 0.03 ± 0.02 3.45 ± 0.58 0.02 ± 0.01 4.33 ± 0.52 AY921992_s 1.65 ± 0.30 0.02 ± 0.01 2.67 ± 0.53 0.02 ± 0.02 0.02 ± 0.01 AM696938_s 5.64 ± 0.85 0.08 ± 0.04 9.40 ± 1.49 0.05 ± 0.03 15.67 ± 2.19 FJ810576_s 2.41 ± 0.82 0.05 ± 0.03 3.51 ± 0.82 0.01 ± 0.01 5.80 ± 1.00 AB672115_s 4.57 ± 0.29 6.62 ± 0.47 4.13 ± 0.36 7.18 ± 0.66 5.69 ± 0.33 EF492955_s 0.80 ± 0.13 1.59 ± 0.19 0.05 ± 0.03 1.37 ± 0.19 1.73 ± 0.27 Clostridium_saccharoperbutylacetonicum 5.34 ± 0.32 9.98 ± 0.82 9.62 ± 0.84 0.13 ± 0.06 0.13 ± 0.05 AB240328_s 1.65 ± 0.36 3.78 ± 0.57 3.53 ± 0.48 3.82 ± 0.42 3.85 ± 0.90 FJ936969_s 0.91 ± 0.26 0.01 ± 0.01 1.43 ± 0.41 0.02 ± 0.01 0.01 ± 0.00 AY221607_s 1.23 ± 0.30 2.45 ± 0.78 2.01 ± 0.65 2.33 ± 0.63 0.03 ± 0.01 FJ793157_s 6.24 ± 0.54 8.53 ± 0.38 8.01 ± 0.28 9.64 ± 1.37 15.72 ± 0.92 AB672259_s 0.96 ± 0.19 2.35 ± 0.16 0.02 ± 0.02 0.01 ± 0.00 0.02 ± 0.01 AY921885_s 2.03 ± 0.48 0.10 ± 0.05 0.07 ± 0.02 4.41 ± 1.25 4.53 ± 0.90 unclassified 2.11 ± 0.67 3.31 ± 0.55 1.20 ± 0.77 0.44 ± 0.25 0.60 ± 0.28

상기 표 5로부터 혼합공간서열을 포함한 프라이머를 사용하는 것이 미생물 군집체 분석에 유용함을 확인할 수 있다.From Table 5 it can be seen that the use of a primer containing a mixed spatial sequence is useful for microbial colony analysis.

이상에서는 본 발명의 바람직한 실시예에 대해서 설명하였으나, 본 발명은 상술한 특정의 실시예에 한정되지 아니하며, 당해 기술분야에서 통상의 지식을 가진 자라면 본원 발명의 요지를 벗어남이 없이 다양한 변형 실시가 가능함은 물론이다. 따라서, 본 발명의 범위는 위의 실시예에 국한해서 해석되어서는 안되며, 후술하는 특허청구범위 뿐만 아니라 이 특허청구범위와 균등한 것들에 의해 정해져야 할 것이다.In the above description of the preferred embodiment of the present invention, the present invention is not limited to the specific embodiments described above, those skilled in the art various modifications without departing from the gist of the present invention Of course it is possible. Therefore, the scope of the present invention should not be construed as being limited to the above embodiments, but should be defined by the claims below and equivalents thereof.

본 융합 프라이머를 활용하여 미생물 군집 분석을 이용한 학술 조사나 품질 관리에 활용하거나 시료내 특정 유전자의 상대적인 양을 측정하는데 사용될 수 있다.The fusion primers can be used for academic research or quality control using microbial community analysis or to measure the relative amounts of specific genes in a sample.

서열번호 01 : AATGATACGGCGACCACCGAGATCTACACSEQ ID NO: 01 AATGATACGGCGACCACCGAGATCTACAC

서열번호 02 : CAAGCAGAAGACGGCATACGAGATSEQ ID NO: 02 CAAGCAGAAGACGGCATACGAGAT

서열번호 03 : AGAGTAGASEQ ID NO: 03 AGAGTAGA

서열번호 04 : AAGGAGTASEQ ID NO: 04: AAGGAGTA

서열번호 05 : CTCTCTATSEQ ID NO: 05 CTCTCTAT

서열번호 06 : CTAAGCCTSEQ ID NO: 06: CTAAGCCT

서열번호 07 : GTAAGGAGSEQ ID NO: 07: GTAAGGAG

서열번호 08 : TATCCTCTSEQ ID NO: 08 TATCCTCT

서열번호 09 : TAGATCGCSEQ ID NO: 09 TAGATCGC

서열번호 10 : TATCCTCT SEQ ID NO: 10 TATCCTCT

서열번호 11 : CAGCCTCGSEQ ID NO: 11 CAGCCTCG

서열번호 12 : TTCTGCCTSEQ ID NO: 12: TTCTGCCT

서열번호 13 : CCTCTCTGSEQ ID NO: 13 CCTCTCTG

서열번호 14 : TCCTCTACSEQ ID NO: 14: TCCTCTAC

서열번호 15 : CATGCCTASEQ ID NO: 15 CATGCCTA

서열번호 16 : TGCCTCTTSEQ ID NO: 16: TGCCTCTT

서열번호 17 : TCGCCTTASEQ ID NO: 17: TCGCCTTA

서열번호 18 : AGCGTAGCSEQ ID NO: 18: AGCGTAGC

서열번호 19 : GTAGAGAGSEQ ID NO: 19: GTAGAGAG

서열번호 20 : CTAGTACGSEQ ID NO: 20 CTAGTACG

서열번호 21 : AGGAGTCCSEQ ID NO: 21 AGGAGTCC

서열번호 22 : GCTCAGGASEQ ID NO: 22 GCTCAGGA

Claims (18)

플로우셀 (flow cell)에 고정된 전방 올리고(oligo)와 결합하는 제 1 전방 어댑터, 전방 시퀀싱 프라이머와 결합하는 제 2 전방 어댑터, 및 목표 유전자의 전방 유전자와 결합하는 전방 유전자 특이적 서열을 나열된 순으로 포함하는 전방 프라이머; 및 The first forward adapter that binds to the front oligo immobilized on the flow cell, the second forward adapter that binds to the forward sequencing primer, and the forward gene specific sequence that binds to the forward gene of the target gene are listed in order. Front primer comprising; And 플로우셀 (flow cell)에 고정된 후방 올리고(oligo)와 결합하는 제 1 후방 어댑터, 후방 시퀀싱 프라이머와 결합하는 제 2 후방 어댑터, 및 목표 유전자의 후방 유전자와 결합하는 후방 유전자 특이적 서열을 나열된 순으로 포함하는 후방 프라이머를 포함하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.The first rear adapter that binds to the rear oligo immobilized on the flow cell, the second rear adapter that binds to the rear sequencing primer, and the rear gene specific sequence that binds to the rear gene of the target gene A primer set for a library for sequencing, comprising a back primer comprising a. 청구항 1에 있어서, The method according to claim 1, 상기 제 2 전방 어댑터와 전방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 전방 혼합공간서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.A primer set for sequencing, characterized in that it further comprises a front mixed space sequence for improving sequence diversity between the second forward adapter and the forward gene specific sequence. 청구항 1에 있어서, The method according to claim 1, 상기 제 2 후방 어댑터와 후방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 후방 혼합공간서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.A primer set for sequencing the library, characterized in that it further comprises a rear mixing space sequence for improving sequence diversity between the second rear adapter and the rear gene specific sequence. 청구항 1에 있어서, The method according to claim 1, 상기 제 1 전방 어댑터와 전방 유전자 특이적 서열 사이에, 샘플의 유래 확인을 용이하게 하는 전방 표지인식서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.Between the first anterior adapter and anterior gene specific sequence, further comprising a front label identification sequence to facilitate the origin of the sample, primer set for sequencing library. 청구항 1에 있어서, The method according to claim 1, 상기 제 1 후방 어댑터와 후방 유전자 특이적 서열 사이에, 샘플의 유래 확인을 용이하게 하는 후방 표지인식서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.Between the first rear adapter and the rear gene specific sequence, further comprises a rear label recognition sequence to facilitate the origin of the sample, primer set for sequencing library. 청구항 2에 있어서, The method according to claim 2, 상기 전방 혼합공간서열과 전방 유전자 특이적 서열 사이에 전방 링커를 추가로 포함하는 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.A primer set for sequencing, further comprising a front linker between the front mixed space sequence and the front gene specific sequence. 청구항 3에 있어서, The method according to claim 3, 상기 후방 혼합공간서열과 후방 유전자 특이적 서열 사이에 후방 링커를 추가로 포함하는 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.And a rear linker between the rear mixed space sequence and the rear gene specific sequence. 청구항 1에 있어서, The method according to claim 1, 상기 제 1 전방 어댑터는 서열번호 1의 서열인 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.The first front adapter is a sequence of SEQ ID NO: 1, characterized in that the primer set for sequencing library. 청구항 1에 있어서, The method according to claim 1, 상기 제 1 후방 어댑터는 서열번호 2의 서열인 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.The first rear adapter is a sequence of SEQ ID NO: 2, characterized in that the primer set for sequencing library. 청구항 1에 있어서, The method according to claim 1, 상기 전방 표지인식서열은 서열번호 3 내지 10 중 어느 한 서열인 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.The front label recognition sequence is characterized in that any one of SEQ ID NO: 3 to 10, a primer set for a sequencing library. 청구항 1에 있어서, The method according to claim 1, 상기 후방 표지인식서열은 서열번호 11 내지 22 중 어느 한 서열인 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.The rear label recognition sequence is characterized in that any one of SEQ ID NO: 11 to 22, primer set for a library for sequencing. 청구항 2 또는 청구항 4에 있어서, The method according to claim 2 or 4, 상기 상기 전방 혼합공간서열 또는 후방 혼합공간서열을 구성하는 염기는 The base constituting the front mixed space sequence or the rear mixed space sequence is (조건 1) 전방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 23개이고, 후방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 15임, 또는 (Condition 1) the base constituting the front mixed space sequence is absent or 1 to 23, and the base constituting the rear mixed space sequence is absent or 1 to 15, or (조건 2) 전방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 15개이고, 후방 혼합공간서열을 구성하는 염기는 부재하거나 1 내지 23임을 만족하는 것을 특징으로 하는, 염기서열분석을 위한 라이브러리용 프라이머 세트.(Condition 2) A primer for sequencing, characterized in that the base constituting the front mixed space sequence is absent or 1 to 15, and the base constituting the rear mixed space sequence is absent or 1 to 23 is satisfied. set. (A) 플로우셀 (flow cell)에 고정된 전방 올리고(oligo)와 결합하는 제 1 전방 어댑터, 전방 시퀀싱 프라이머와 결합하는 제 2 전방 어댑터, 및 목표 유전자의 전방 유전자와 결합하는 전방 유전자 특이적 서열을 나열된 순으로 포함하는 전방 프라이머; 및 플로우셀 (flow cell)에 고정된 후방 올리고(oligo)와 결합하는 제 1 후방 어댑터, 후방 시퀀싱 프라이머와 결합하는 제 2 후방 어댑터, 및 목표 유전자의 후방 유전자와 결합하는 후방 유전자 특이적 서열을 나열된 순으로 포함하는 후방 프라이머를 분석하고자 하는 염기서열의 양단에 결합시키는 단계, (A) a first anterior adapter that binds an anterior oligo immobilized to a flow cell, a second anterior adapter that binds an anterior sequencing primer, and an anterior gene specific sequence that binds an anterior gene of a target gene A front primer comprising in the order listed; And a first rear adapter that binds to a posterior oligo immobilized on a flow cell, a second rear adapter that binds to a rear sequencing primer, and a rear gene specific sequence that binds to the rear gene of a target gene. Binding the rear primer including the sequence to both ends of the base sequence to be analyzed; (B) 상기 전방 프라이머 및 후방 프라이머가 양단에 결합된 염기서열을 연쇄중합효소반응으로 증폭시키는 단계, 및 (B) amplifying a nucleotide sequence in which the front and rear primers are coupled at both ends by a chain polymerase reaction, and (C) 상기 증폭된 염기서열을 정제하는 단계를 포함하는, 염기서열분석용 라이브러리의 제조방법.(C) a method of preparing a library for sequencing, comprising the step of purifying the amplified base sequence. 청구항 13에 있어서, The method according to claim 13, 상기 제 2 전방 어댑터와 전방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 전방 혼합공간서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석용 라이브러리의 제조방법.Method for producing a sequencing library characterized in that it further comprises a front mixed space sequence for improving sequence diversity between the second front adapter and the front gene-specific sequence. 청구항 13에 있어서, The method according to claim 13, 상기 제 2 후방 어댑터와 후방 유전자 특이적 서열 사이에 서열 다양성 향상을 위한 후방 혼합공간서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석용 라이브러리의 제조방법.And a rear mixed space sequence for improving sequence diversity between the second rear adapter and the rear gene specific sequence. 청구항 13에 있어서, The method according to claim 13, 상기 제 1 전방 어댑터와 전방 유전자 특이적 서열 사이에, 샘플의 유래 확인을 용이하게 하는 전방 표지인식서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석용 라이브러리의 제조방법.Between the first anterior adapter and anterior gene specific sequence, further comprising an anterior label recognition sequence for facilitating the origin of the sample, the method for producing a sequencing library. 청구항 13에 있어서, The method according to claim 13, 상기 제 1 후방 어댑터와 후방 유전자 특이적 서열 사이에, 샘플의 유래 확인을 용이하게 하는 후방 표지인식서열을 추가로 포함하는 것을 특징으로 하는, 염기서열분석용 라이브러리의 제조방법.Between the first rear adapter and the rear gene specific sequence, further comprising a rear label recognition sequence to facilitate the origin of the sample, characterized in that the method for producing a sequencing library. 청구항 13에 있어서, The method according to claim 13, 상기 증폭시키는 단계 및 정제하는 단계는 각각 1 회씩만 포함되는, 염기서열분석용 라이브러리의 제조방법.The amplifying and purifying steps are included only once each, method for preparing a sequencing library.
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