WO2019085546A1 - Procédé de construction d'une banque pour séquençage de molécules uniques d'adnr 16s microbien - Google Patents

Procédé de construction d'une banque pour séquençage de molécules uniques d'adnr 16s microbien Download PDF

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WO2019085546A1
WO2019085546A1 PCT/CN2018/095582 CN2018095582W WO2019085546A1 WO 2019085546 A1 WO2019085546 A1 WO 2019085546A1 CN 2018095582 W CN2018095582 W CN 2018095582W WO 2019085546 A1 WO2019085546 A1 WO 2019085546A1
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Prior art keywords
sequencing
primer
amplification
rdna
microbial
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PCT/CN2018/095582
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English (en)
Chinese (zh)
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林东旭
刘小龙
杨明燕
魏国鹏
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南京格致基因生物科技有限公司
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Publication of WO2019085546A1 publication Critical patent/WO2019085546A1/fr

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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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to the field of microbial gene sequencing, in particular to a method for constructing an amplification primer and a sequencing library for sequencing 16S rDNA single molecule level of bacteria and archaea.
  • the existing single-molecule 16S rDNA sequencing technology mainly uses the sequencing platform of ion torrent.
  • the sequencing principle of ion torrent is to amplify a single template by micro-oil droplets, so it can achieve single molecule level.
  • the currently more widely used illumina sequencing platform cannot achieve 16S rDNA single-molecule sequencing analysis.
  • the technical problem to be solved by the present invention is to provide a method for constructing a microbial 16S rDNA single molecule horizontal sequencing library with low cost and simple operation.
  • a second primer pair for library construction contains P1 and P2 short primers.
  • the sequences of the upstream primer P1 and the downstream primer P2 are set forth in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
  • the invention provides a method of constructing a 16S rDNA hypervariable region sequencing library of a microorganism, the method comprising: amplifying a microbial bacterial genome 16S rDNA using the primer pair of the invention to construct a sequencing library.
  • the method of constructing a 16S rDNA sequencing library of microbial bacteria comprises the following steps:
  • the sample in step a) is a stool sample.
  • the concentration and purity are measured using a micro-ultraviolet spectrophotometer after extraction of the sample metagenomic DNA.
  • the amplification in step b) is a two-step amplification
  • the first step using the first primer pair of the present invention to amplify two cycles using the sample metagenomic DNA as a template to ensure that the two segments are connected with a linker.
  • a complete and single library fragment was generated;
  • the second step amplification was performed using the first amplified product as a template, and the second primer pair of the present invention was used to amplify for 30 cycles to amplify the signal.
  • the product obtained after two-step amplification is subjected to agarose gel electrophoresis.
  • purification is carried out in step c) using AMPure XP magnetic beads from Beckman & Coulter.
  • the purified sequencing library is subjected to accurate quantification and fragment size analysis using Qubit 3.0 and Agilent 2100, and the sequencing library is diluted according to the sequencing platform requirements, and the results of Qubit 3.0 quantification are used as standards and mixed according to the amount of data required. Finally, sequencing was performed using Illumina HiseqX or Miseq.
  • the method for constructing the 16S rDNA single molecule level sequencing library of the microorganism of the present invention is different from the prior art in that:
  • the primer designed by the invention is a long primer containing a UID random tag, and the two-step amplification can ensure that one label corresponds to the unique amplified 16S rDNA template, and finally the single molecule template can be accurately determined in the analysis of the sequencing result.
  • the level of the 16S rDNA flora abundance and composition can be calculated more accurately.
  • the 16S rDNA template of the sample to be tested is subjected to the first step of amplification, and the two ends are labeled with a unique label, and the second step amplifies the effective amplification signal but No increase in the number of tags for subsequent sequencing and analysis.
  • the invention realizes the sequencing and analysis of the single molecule 16S rDNA through the illumina sequencing platform, breaks the original situation that the single molecule 16S rDNA can only be analyzed through the ion torrent sequencing platform, simplifies the experimental process and reduces the sequencing cost.
  • the target fragment amplified by the long primer provided by the invention is the V3-V4 region of 16S rDNA, and the amplified fragment is longer in length than the single segment of 16S rDNA only, and contains more information and distinguishes. Higher rate, easy to distinguish between species
  • N is an uncertain base and N is selected from A, C, G, and T Any one of them; index: split tag sequence; UID: random tag; 16S rDNA primer: 16S rDNA primer sequence;
  • FIG. 2 is an agarose gel electrophoresis pattern of a two-step amplification method using a 16S rDNA single-molecule level sequencing library of the microorganism of the present invention, wherein the left side is a product electrophoresis pattern after two-step amplification, and the DNA marker is on the right side. ;
  • Figure 3 is a 2100 quality control peak map of a 16S rDNA single molecule level sequencing library after magnetic bead purification
  • nucleic acid refers to a polymer of nucleotides, including any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), a DNA representation of mRNA, usually reversed by messenger RNA (mRNA). Recorded, or obtained by amplification; synthetically produced or amplified by production of DNA molecules; and mRNA.
  • genomic DNA complementary DNA
  • cDNA complementary DNA
  • mRNA messenger RNA
  • oligonucleotide refers to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more specifically shorter than 100 nucleotides, and most particularly shorter than 50 nucleosides. acid. Typically, an oligonucleotide is a single stranded DNA molecule.
  • the term "primer” refers to an oligonucleotide under suitable conditions (ie, in the presence of four different nucleoside triphosphates and a polymerization reagent (eg, DNA or RNA polymerase or reverse transcriptase)
  • a polymerization reagent eg, DNA or RNA polymerase or reverse transcriptase
  • the oligonucleotide is capable of hybridizing to a nucleic acid (also referred to as "annealing") in a suitable buffer and at a suitable temperature and is used as a starting point for nucleotide (RNA or DNA) polymerization.
  • primer length refers to a portion of an oligonucleotide or nucleic acid that hybridizes to a complementary "target" sequence and initiates nucleotide synthesis. Short primer molecules generally require cooler temperatures to form a sufficiently stable hybrid complex with the template. Primers do not have to reflect the exact sequence of the template but must be sufficiently complementary to hybridize to the template.
  • Reagent broadly refers to any reagent used in the reaction other than an analyte, such as a nucleic acid being analyzed.
  • Illustrative reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, and the like.
  • Reagents for the enzymatic reaction include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.
  • the method for constructing the 16S rDNA single molecule level sequencing library of the microorganism of the present embodiment is carried out as follows:
  • the first step of amplification was carried out using Invitrogen's multiplex amplification reagent (Invitrogen's Multiplex PCR master mix); the first step of amplification of 23 ⁇ L PCR reaction system is shown in Table 1, the first step of the amplification reaction procedure as As shown in Table 2, the primer sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2; as shown in FIG. 1;
  • Composition Volume/DNA volume First step amplification system 23uL Multiplex PCR master mix 25uL Upstream primer P1 at a concentration of 10uM 1uL Downstream primer P2 with a concentration of 10uM 1uL
  • Fecal samples stored in 1mL of fecal preservation solution (can be stored at room temperature for 15 days), using Tianmu's fecal microbial DNA extraction kit (ZYMO Quick-DNA TM Fecal Microbe Microprep Kit), according to the instructions
  • Tianmu's fecal microbial DNA extraction kit ZYMO Quick-DNA TM Fecal Microbe Microprep Kit
  • the steps of extracting fecal macrogenomic DNA; genital secretion samples, stored in 1mL preservation solution and mixed can be stored at room temperature for 15 days
  • Kaijie's DNA extraction kit Qiagen's QIAamp DNA Mini Kit
  • Bioinformatics analysis was performed on the sequencing results, and all the reads with repeated tags and repeated sequences at both ends were combined.
  • the single-ended tags were not duplicated after the combination, and the one-step amplification and sequencing results were not tagged.
  • the microbial composition of the female reproductive tract is relatively simple, and the lactic acid bacteria account for 99%.
  • the composition and proportion of intestinal microflora are diversified. It can be seen from the figure that the actual proportion of some microflora is exaggerated, such as faecalibacterium, phascolarctobacterium, which actually accounts for only 1%, and the conventional one-step amplification has a preference. Zoom in to 17%. The same one-step amplification of 4% of Blautia is underestimated, in fact, 11%. Therefore, it can be concluded that the single-molecule two-step method is more accurate for the evaluation of the proportion of microbial populations than the traditional one-step method.

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Abstract

L'invention concerne un procédé de construction d'une banque pour séquençage de molécules uniques d'ADNr 16S microbien, se rapportant au domaine technique de la biochimie. Le procédé de construction de banque comprend les étapes suivantes : collecte d'échantillons et extraction d'ADN; amplification; purification; quantification; et séquençage et analyse bioinformatique. Le procédé de construction d'une banque pour séquençage de molécules uniques d'ADNr 16S microbien selon l'invention est peu coûteux et simple de fonctionnement, et peut simplifier les procédés expérimentaux et réduire le coût de séquençage.
PCT/CN2018/095582 2017-10-31 2018-07-13 Procédé de construction d'une banque pour séquençage de molécules uniques d'adnr 16s microbien WO2019085546A1 (fr)

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CN201711049666.6A CN108070643A (zh) 2017-10-31 2017-10-31 微生物16S rDNA单分子水平测序文库的构建方法

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CN108070643A (zh) * 2017-10-31 2018-05-25 南京格致基因生物科技有限公司 微生物16S rDNA单分子水平测序文库的构建方法
CN108866220B (zh) * 2018-08-15 2022-03-04 博奥生物集团有限公司 一种用于鼻腔菌群检测的方法及检测试剂盒
WO2020164015A1 (fr) * 2019-02-13 2020-08-20 武汉华大医学检验所有限公司 Amorce de fusion pour construction de banque de séquençage de troisième génération, et procédé de construction de banque, procédé de séquençage et kit de construction de banque correspondant
CN113025761A (zh) * 2021-05-27 2021-06-25 广州赛哲生物科技股份有限公司 病原微生物鉴定的多重扩增配合高通量测序方法及试剂盒

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102409049A (zh) * 2010-09-21 2012-04-11 深圳华大基因科技有限公司 一种基于pcr的dna标签文库构建方法
CN103045726A (zh) * 2012-11-20 2013-04-17 南方科技大学 对多个混合dna或rna序列进行基因测序的方法及装置
CN104153004A (zh) * 2014-08-11 2014-11-19 上海美吉生物医药科技有限公司 一种用于扩增子测序的建库方法
WO2015097006A1 (fr) * 2013-12-24 2015-07-02 Universite De Liege Analyse métagénomique d'échantillons
WO2015187953A2 (fr) * 2014-06-04 2015-12-10 Quest Diagnostics Investments Incorporated Méthode d'identification microbienne directe
CN106591458A (zh) * 2016-12-23 2017-04-26 深圳市前海金卓生物技术有限公司 生物饲料活性度的评价方法
CN107267435A (zh) * 2017-05-26 2017-10-20 中国科学院南京土壤研究所 一种溴代烃厌氧降解菌剂的制备方法及应用
CN107937582A (zh) * 2017-12-29 2018-04-20 苏州普瑞森基因科技有限公司 一种用于分析肠道微生物的引物组及其应用
CN108070643A (zh) * 2017-10-31 2018-05-25 南京格致基因生物科技有限公司 微生物16S rDNA单分子水平测序文库的构建方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106497926A (zh) * 2016-11-03 2017-03-15 承启医学（深圳）科技有限公司 一种用于构建微生物细菌16s　rDNA可变区测序文库的扩增子引物及构建方法

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102409049A (zh) * 2010-09-21 2012-04-11 深圳华大基因科技有限公司 一种基于pcr的dna标签文库构建方法
CN103045726A (zh) * 2012-11-20 2013-04-17 南方科技大学 对多个混合dna或rna序列进行基因测序的方法及装置
WO2015097006A1 (fr) * 2013-12-24 2015-07-02 Universite De Liege Analyse métagénomique d'échantillons
WO2015187953A2 (fr) * 2014-06-04 2015-12-10 Quest Diagnostics Investments Incorporated Méthode d'identification microbienne directe
CN104153004A (zh) * 2014-08-11 2014-11-19 上海美吉生物医药科技有限公司 一种用于扩增子测序的建库方法
CN106591458A (zh) * 2016-12-23 2017-04-26 深圳市前海金卓生物技术有限公司 生物饲料活性度的评价方法
CN107267435A (zh) * 2017-05-26 2017-10-20 中国科学院南京土壤研究所 一种溴代烃厌氧降解菌剂的制备方法及应用
CN108070643A (zh) * 2017-10-31 2018-05-25 南京格致基因生物科技有限公司 微生物16S rDNA单分子水平测序文库的构建方法
CN107937582A (zh) * 2017-12-29 2018-04-20 苏州普瑞森基因科技有限公司 一种用于分析肠道微生物的引物组及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
D'AMORE, R. ET AL.: "A Comprehensive Benchmarking Study of Protocols and Sequencing Platforms for 16S rRNA Community Profiling", BMC GENOMIC, vol. 17, no. 55, 14 January 2016 (2016-01-14), pages 1 - 20, XP021231629, DOI: 10.1186/s12864-015-2194-9 *
LI, ZONGZE: "Oral Microoriganisms Metagenomics Research in Patients with Type II Diabetes", CHINA MASTER'S THESES FULL-TEXT DATABASE, 15 March 2018 (2018-03-15), pages 23 - 26 *
ZONGZE ET AL.: "Analysis of Oral Microbial Diversity in Patients with Type 2 Diabetes Mellitus by 16s Ribosome DNA Amplicon Sequencing", TRANSLATIONAL MEDICINE JOURNAL, vol. 6, no. 3, 30 June 2017 (2017-06-30), pages 151 - 156 *

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