WO2021203461A1 - Système de code à barres d'ancrage de position pour construction de banque de séquençage de nanopores - Google Patents

Système de code à barres d'ancrage de position pour construction de banque de séquençage de nanopores Download PDF

Info

Publication number
WO2021203461A1
WO2021203461A1 PCT/CN2020/085645 CN2020085645W WO2021203461A1 WO 2021203461 A1 WO2021203461 A1 WO 2021203461A1 CN 2020085645 W CN2020085645 W CN 2020085645W WO 2021203461 A1 WO2021203461 A1 WO 2021203461A1
Authority
WO
WIPO (PCT)
Prior art keywords
barcode
sequence
anchor
anchored
sequencing
Prior art date
Application number
PCT/CN2020/085645
Other languages
English (en)
Chinese (zh)
Inventor
戴岩
胡龙
张烨
肖念清
任用
Original Assignee
江苏先声医学诊断有限公司
北京先声医学检验实验室有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏先声医学诊断有限公司, 北京先声医学检验实验室有限公司 filed Critical 江苏先声医学诊断有限公司
Publication of WO2021203461A1 publication Critical patent/WO2021203461A1/fr

Links

Images

Classifications

    • 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
    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B80/00—Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Definitions

  • the invention relates to the field of gene sequencing, in particular to a position-anchored barcode system for nanopore sequencing database construction.
  • Illumina second-generation sequencing is in full swing in China, but there are the following problems when applied to microbial detection: First, the read length of second-generation sequencing is less than a few hundred bp, and there will be high homologous sequences between different species of microorganisms, resulting in The accuracy of metagenomic species analysis is poor, and irrelevant microbial information is fed back in the data report, which causes greater diagnostic interference for doctors; secondly, the identification of deeper disease-causing genes and drug-resistant genes requires assembly and splicing of sequencing sequences Therefore, complex analysis requires higher time and capital costs to make up for the read length defect of the second-generation sequencing data; in addition, the second-generation sequencing-related instruments are expensive, cumbersome to operate, high initial investment, and the entire sequencing time is long, which is difficult.
  • the third-generation sequencing technology PacBio has greatly improved the read length of sequencing, and can detect long fragment data of 8-12kb, or even 40-70kb, but its disadvantage is that the library construction process is more complicated. Moreover, like the second-generation sequencing, there is the disadvantage of a long sequencing cycle. After a round of sequencing, it takes tens of hours to complete the data offline. With the subsequent analysis time, it is difficult to meet the rapid identification of pathogenic microorganisms.
  • Nanopore sequencing technology just makes up for the disadvantages of other sequencing platforms, not only reading long sequence fragments, but also short library building and sequencing time.
  • the equipment is small and portable, and the data generation and bio-information analysis can be real-time, which perfectly solves the limitation of the sequencing site and the delay of report feedback. Therefore, this technology is very suitable for the analysis and identification of clinical infection microbial pathogens.
  • the on-board chip for nanopore sequencing is very expensive and the price is very unfriendly to users.
  • Using barcode information to distinguish multiple samples is a common cost-saving strategy for high-throughput DNA sequencing. For each DNA sample, a unique barcode sequence is introduced during the library building process.
  • the sequencing reads are classified according to the barcode sequence to distinguish different samples on the computer.
  • the expensive chip in the nanopore sequencing technology makes multiple samples on the machine have obvious economic advantages, allowing users to share the fixed cost of a flow cell.
  • a series of kits launched by Oxford Nanopore Company provide 12 different barcodes with a length of 24bp. These barcodes are connected to the two ends of the sample DNA sequence during the library construction process and then sequenced on the computer, so that a chip can obtain 12 different samples at the same time. Sequence information. However, when distinguishing samples based on the barcode sequence later, it was found that the 24bp long barcode that comes with the library building kit is seriously confused.
  • the technical problem to be solved by the present invention is to improve the accuracy of the barcode comparison process of the existing nanopore sequencing data sample.
  • the invention categorizes and statistically analyzes the errors that occur during sequence comparison of the nanopore sequencing platform by digging in-depth large amounts of data, and quantifies the influence of different error rate types on sequence identification.
  • the sequencing error of the indel type (Indel) will greatly increase the error rate of sequence identification, while the base mismatch type (Mismatch) has little effect on the improvement of the error rate of sequence identification, so it is blindly improved in the design of sample barcodes.
  • the length of the barcode has a limited effect on the accuracy improvement, and adding a position anchor sequence to the barcode of the same length improves the accuracy even more.
  • the present invention constructs a set of "position-anchored barcode system” containing position-anchored sequences, and validates it based on Nanopore’s library building kit SQK-PBK004, and builds a library of 10 pure bacteria on the computer.
  • the original barcode system and the position-anchored barcode system are used to classify and compare the off-machine data.
  • the results show that the position-anchored barcode system has better sample classification accuracy, which is more than 3 orders of magnitude higher than the original barcode system.
  • the first object of the present invention is to provide a position-anchored barcode system that improves the resolution accuracy of sample nanopore sequencing.
  • the second object of the present invention is to provide a preparation method and application of the above-mentioned position-anchored barcode system.
  • the present invention provides a position-anchored barcode system for nanopore sequencing library construction, which is characterized in that the system includes the following structure:
  • the BARCODE is a barcode sequence
  • the ANCHOR is an anchor sequence.
  • the system includes the following structure: FLANK1-[BARCODE-ANCHOR] n -BARCODE n+1 -FLANK2,
  • the FLANK is a flanking sequence
  • the BARCODE sequences are the same or different; preferably, the BARCODE sequences are different;
  • the ANCHOR sequences are the same or different; preferably, the ANCHOR sequences are different.
  • the length of the ANCHOR sequence is 5-50 bp; preferably, the length of the ANCHOR sequence is 10-35 bp;
  • the homology between the ANCHOR sequence and the BARCODE sequence is ⁇ 70%; preferably ⁇ 50%.
  • the length of the FLANK is 10-30 bp; preferably, the length of the FLANK sequence is 15-25 bp;
  • the position-anchored barcode system for nanopore sequencing library construction is characterized in that the system includes any of the following structures:
  • the ANCHOR sequences are different or the same, preferably the ANCHOR sequences are different;
  • the BARCODE sequences are different or the same, preferably the BARCODE sequences are different.
  • the present invention also provides a method for preparing the above-mentioned position-anchored barcode system for nanopore sequencing library construction, characterized in that: the method includes directly synthesizing the nucleotide sequence of the position-anchored barcode system, or through analysis After the segments are synthesized, they are connected to prepare the position-anchored barcode system.
  • the preparation method is as follows: on the basis of the existing nanopore sequencing library barcode, bridge primers are used to realize the existing barcode adapter and The series of barcode linkers is designed; preferably, the bridging primer sequence is ANCHOR in the structure of the position-anchored barcode system.
  • the existing barcode connector is derived from the original barcode of the SQK-PBK004 kit of ONT.
  • the present invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library building in improving the accuracy of sequencing samples classification.
  • the present invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library building in reducing false positives of sequencing samples.
  • the invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library construction in sequencing library construction.
  • the present invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library construction in sequencing.
  • the present invention also provides a method for constructing a sequencing library, which is characterized in that the position-anchored barcode system of the above-mentioned nanopore sequencing library is used to construct a sequencing library.
  • the present invention also provides a sequencing adapter, characterized in that the sequence of the sequencing adapter includes the position anchor barcode system described above.
  • the present invention also provides a composite, characterized in that the composition is connected to the above-mentioned position-anchored barcode system.
  • the present invention also provides a composition, characterized in that the composition contains the above-mentioned position-anchored barcode system.
  • the present invention also provides a kit for nanopore sequencing library construction, characterized in that the kit contains the above-mentioned position-anchored barcode system or the above-mentioned sequencing adapter.
  • the present invention proves for the first time that indel type errors are the main reason for overall sequence alignment errors. In contrast, base mismatch types have less impact on overall sequence alignment errors.
  • the present invention restricts indel type errors from extending to the overall comparison result by introducing an anchor sequence in the barcode system, greatly reduces the degradation of the comparison score caused by indels, and screens out long-distance barcode interference to achieve Accurate bar code resolution; compared to only increasing the length of the bar code sequence, although increasing the bar code length can appropriately reduce sample classification errors caused by base mismatches, the accuracy of the overall sequence comparison result is very limited.
  • the position-anchored barcode system has an extremely significant effect on improving the accuracy of the results.
  • the present invention is based on the library building process of the nanopore platform SQK-PBK004, cleverly uses its built-in barcode to connect the independently developed barcode sequence, and uses the connecting part sequence as the anchor sequence to design FLANK1-BARCODE 1 -ANCHOR 2 -BARCODE 2- FLANK2 type position anchoring barcode system, this system can improve the classification accuracy from 0.999 to 0.999999 when distinguishing different samples.
  • the position-anchored barcode system of the present invention can design barcodes of different lengths and the number of anchor sequences according to different requirements, so as to achieve the balance of classification accuracy and microbial detection rate for different requirements.
  • the position-anchored barcode system of the present invention has better resolution, higher accuracy, and reduced false positive identification, can improve the accuracy of nanopore sequencing as a whole, reduce sequencing costs, and is suitable for popularization and use.
  • Figure 1 Error rate statistics based on the sequencing data of the kit barcode system;
  • Figure A shows the average error rate and median error rate of each site in the actual sequencing of the barcode adapter sequence of the 10 sets of kits;
  • Figure B shows the barcode of the 10 sets of kits In the actual sequencing of the adapter sequence, the average error rate and the median error rate of the three types of errors of insertion, deletion, and mismatch at each site are aligned;
  • Figure C shows the corresponding relationship between the barcode adapter site of the kit and the alignment error;
  • Figure D shows A summary of the subcategories of the alignment error types of the barcode adapter in the kit, which shows the distribution of alignment errors that occur at different sites.
  • the abscissa is the base position of the barcode sequence, and the ordinate is the error type.
  • the color of the small cells in the figure indicates that The error rate of the error type at the site, the darker the color means the higher the error rate.
  • the color blocks in the Block comment area indicate different components of the connector sequence, and the error type is clustered by Euclidean distance;
  • Figure 2 The influence of different alignment error types on the accuracy of overall sequence classification; Figure A shows the total error rate is 8%; Figure B shows the total error rate is 16%;
  • the terms “including”, “including”, “having”, “containing” or “involving” are inclusive or open-ended, and do not exclude other unlisted elements or method steps .
  • the term “consisting of” is considered a preferred embodiment of the term “comprising”. If in the following a certain group is defined as comprising at least a certain number of embodiments, this should also be understood as revealing a group preferably consisting of only these embodiments.
  • the "position-anchored barcode system" of the present invention refers to a multi-barcode sequencing tag system containing two or more BARCODE sequences in series.
  • the BARCODEs are anchored by a specific ANCHOR sequence.
  • This system can be applied to nanopore sequencing.
  • the construction of sequencing library in can improve the accuracy of sequencing samples classification, and reduce the application of sequencing sample classification false positives.
  • Its specific structure can be the [BARCODE-ANCHOR] n -BARCODE n+1 described in the present invention, where n ⁇ 1, the BARCODE is a barcode sequence, and the ANCHOR is an anchor sequence. It can be understood that any composition, compound, or system including the above structure is within the scope of the present invention.
  • the present invention is explained by taking the barcode of the SQK-PBK004 kit in the prior art as an example, it is only an exemplary description and does not limit the present invention.
  • the present invention has passed specific bio-information theory analysis and wet experiment verification, which proves that any barcode system containing [BARCODE-ANCHOR] n -BARCODE n+1 structure can be used for the construction of sequencing library, which can improve the accuracy of sequencing samples classification , To reduce the false positives of sequencing samples.
  • the structure of the position-anchored barcode system may be as follows: FLANK1-[BARCODE-ANCHOR] n -BARCODE n+1 -FLANK2, the FLANK is a linker sequence, and the linker sequence is a sequencing library
  • FLANK1 and 2 can be the same or different in sequence according to actual needs.
  • the length of the position-anchored barcode system of the present invention is appropriately selected in the field according to actual needs.
  • the n is 1, 2 or 3.
  • BARCODE is used as a marker sequence in sequencing.
  • the sequence can be the same or different; in some preferred embodiments, the BARCODE sequence is different.
  • the ANCHOR sequence is used as an anchor component, and the starting sequence may be the same or different.
  • the ANCHOR sequence is different.
  • the length of the ANCHOR sequence can be a length known in the art, for example, it can be 5-50 bp. In some preferred embodiments, the length of the ANCHOR sequence is 10-35 bp.
  • the ANCHOR sequence is used as the anchor component of BARCODE. Its sequence should be distinguished from the BARCODE sequence. There is no special restriction.
  • the homology between the ANCHOR sequence and the BARCODE sequence can be ⁇ 80%, ⁇ 70%, ⁇ 60%, ⁇ 50%, ⁇ 40%, ⁇ 30%, ⁇ 20%, ⁇ 10%; in some preferred embodiments, the homology is ⁇ 50%.
  • the structure can be specifically as follows:
  • the "barcode connector” in the present invention refers to a complete section containing a barcode sequence and flanking sequences at both ends.
  • the self-designed barcode connector is defined as a BBRCD connector
  • the barcode connector in the original kit SQK-PBK004 is defined as an ABRCD connector.
  • bar code sequence in the present invention refers to a specific sequence of a bar code, which is included in the bar code connector and is a part of the sequence of the bar code connector.
  • the independently designed barcode sequence is defined as BBRCD
  • the barcode sequence in the original kit SQK-PBK004 is defined as ABRCD.
  • anchor sequence in the present invention refers to a nucleotide sequence used to anchor BARCODE. Its length can be any verified length known in the art, for example, it can be 5-50 bp. In the embodiment, it can be 10-35bp; its sequence should be distinguished from the BARCODE sequence, and there is no particular limitation.
  • the homology of ANCHOR sequence and BARCODE sequence can be ⁇ 80%, ⁇ 70%, ⁇ 60%, ⁇ 50%, ⁇ 40 %, ⁇ 30%, ⁇ 20%, ⁇ 10%; in some preferred embodiments, the homology is ⁇ 50%.
  • the ANCHOR sequence mentioned in the embodiment of the present invention includes SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 13, and the like.
  • FLANK refers to the flanking sequences at both ends of the barcode system, which are conventional components of sequencing barcode adapters.
  • FLANK1 in the present invention is a Y-type sequencing adapter that connects the motor protein. Ensure that the DNA passes through the nanopore to achieve normal sequencing;
  • FLANK2 is used to connect the sequence of the sequencing sample, and its length can be any verified length known in the art, for example, it can be 10-30 bp, and in some preferred embodiments, it can be 15-25 bp .
  • the present invention considers that the main reason for the confusion of the barcode is the sequence difference between the sequenced barcode and the preset real barcode, so the difference between the barcode sequence obtained by the sequencing and the real barcode sequence is sorted and sorted first.
  • the present invention uses 10 sets of barcodes of ONT's SQK-PBK004 kit to build a separate library of sample DNA, intercept 250bp of the 5'end of the sequencing data to ensure that the barcode region is included, and then perform a global connection with the corresponding preset barcode adapter. Overlap alignment.
  • the comparison difference of each position of the barcode adapter sequence is summarized, and the error position distribution and error type of the sequencing barcode are counted.
  • the error types are divided into three categories, namely insertion (I), deletion (deletion, D) and base mismatch (mismatch, X).
  • FIG. 1B there is a significant difference between the average and median error rates of each point of the barcode connector, so the present invention further summarizes the alignment errors of different error types at different barcode sequence positions.
  • Figure 1C shows that, except for the high error rate of the first 6 bases at the 5'end caused by the initial instability of sequencing, there is no significant error rate difference in the remaining positions.
  • XN represents all mismatch types except GA and AG mismatches
  • I1 represents a single base randomly inserted
  • I2 and II represent randomly inserted 2, 3 to 5 bases, and 3, 4 respectively.
  • the ratio of 5 bases is 8:1.8:0.2
  • X2,XX indicates that the site randomly introduces unmatched bases and then inserts one or more bases.
  • Example 2 The influence of different error types and barcode length on the overall accuracy of barcode comparison
  • the present invention simulates a total of 6 groups of barcode sequences with lengths of 20bp, 40bp, 60bp, 80bp, 100bp, and 120bp, each of which simulates 12 different barcode elements. .
  • the present invention takes 80bp as an example to illustrate the specific overview of the simulation.
  • the present invention presets 12 ideal barcode sequences, and the sequence information is shown in Table 2:
  • a flow cell has multiple samples on the machine, there will be multiple barcode DNA in the actual off-machine data, and the barcodes between the samples will be confused with each other.
  • the present invention simulates 100,000 joint sequences for each preset barcode under each set of length, and all simulated The sequence is mixed together to simulate the situation that 12 samples are on the machine at the same time.
  • the analog sequence name uses the preset barcode name itself plus a digital number. The data is classified through the same biometric analysis process, and finally the barcode information obtained by the classification is compared with the analog sequence name. Determine whether crosstalk occurs.
  • Example 3 The influence of inserting anchor sequence on the overall accuracy of barcode comparison
  • the present invention intends to modify the barcode fragment in the following two ways, taking 80bp as an example:
  • the position range of the short bar code is locked by the position information obtained in the comparison process of the anchor sequence, and finally the data is classified according to the result of the short bar code combination.
  • this embodiment is taken as an example, based on the original ONT company’s nanopore library PCR barcode kit SQK-PBK004, clever use of its existing single barcode adapter (sequence structure FLANK1-ABRCD -FLANK3'), through the connection reaction, connect it with the barcode adapter (sequence structure FLANK5'-BBRCD-FLANK2) independently designed in the present invention to form the position-anchored barcode system of the present invention: FLANK1-ABRCD-ANCHOR- BBRCD-FLANK2 (where the ANCHOR sequence is the sequence after FLANK3' and FLANK5' are connected by a ligation reaction).
  • FLANK2 continues to connect with the sample DNA of known source through the ligation reaction, so that the final DNA to be sequenced has a position-anchored barcode system.
  • This design allows us to infer the connected barcode sequence based on the results of the known sample DNA, thereby quantifying the classification accuracy of the barcode system.
  • the present invention obtains 10 barcode sequences BBRCD with excellent distinguishability from each other through combinatorial comparison; and then adds a 13bp conservative flanking sequence FLANK to the 5'end.
  • the sequence has good PCR primer characteristics, moderate GC content, no hairpin and dimer structure, etc., and the Y-shaped structure of the original PCR linker is used to avoid the situation where multiple barcodes are connected in series in the connection step; ANCHOR sequence, in this experiment It is also the 3'end sequence (SEQ ID NO.13) of the PCR bypass primer, which is consistent with the self-designed barcode adapter FLANK5', and the 5'end base sequence is consistent with the FLANK3' of the original barcode adapter of the kit, thus achieving a
  • the PCR reaction obtains a sequenced DNA fragment with 5'and 3'ends in series with double barcode adapters simultaneously.
  • the PCR bridging primer sequence is as follows:
  • the underline indicates the 13bp sequence consistent with the self-designed barcode linker FLANK5'.
  • This example is aimed at 10 cases of standard pure bacteria strains Brevibacillus borstelensis, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus; Acinetobacter baumannii, Stemallophila subtilis, and the library optimization diagrams used in the construction process
  • Each pure bacteria sample introduces a different position-anchored barcode sequence for library preparation, and specifically prepares 10 sets of position-anchored barcode sequences, as shown in Table 5.
  • Example 4 Based on the experimental procedure of Example 4, single-sample sequencing was performed on 10 different strains, and it was ensured that each sample was connected to only one barcode. Since only a single barcode is used in each sequencing, if the barcode of the corresponding sample is compared with the barcode that is not corresponding to the sample, it is considered to be a misclassification.
  • the biometric analysis of the present invention uses the official software guppy of Oxford Nanopore Company to evaluate the accuracy of sample classification of the original barcode system, and uses an independent software process to evaluate the accuracy of sample classification of the position-anchored barcode system.
  • the present invention examines the resolving power of 10 groups of position-anchored bar codes, and the final accuracy rate of read classification is statistically calculated within the range of these 10 groups of bar codes.
  • the classification accuracy of the position-anchored barcode system reaches 99.9999% on average, as shown in Figure 5.
  • the reads classified into barcode01, barcode02, barcode05, barcode09 and barcode10 are consistent with the classification accuracy of the simulated data, which are all 100%.
  • guppy's 99.9% resolution accuracy it has increased by 3 orders of magnitude, which means that the base for accurately distinguishing samples has increased from a thousand to a million for a single barcode, and the false positive rate caused by misclassification of reads has been reduced by 1000 times.
  • the classification accuracy of position-anchored barcodes is significantly better than that of single barcodes.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un système de code à barres d'ancrage de position pour la construction de banque de séquençage de nanopores, un procédé de préparation et une utilisation du système. Le système de code à barres d'ancrage de position a une résolution supérieure et une précision de classification plus élevée et peut réduire remarquablement l'identification de faux taux positifs, améliorer la précision de séquençage de nanopore global et réduire le coût de séquençage.
PCT/CN2020/085645 2020-04-09 2020-04-20 Système de code à barres d'ancrage de position pour construction de banque de séquençage de nanopores WO2021203461A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010276679.2A CN111440846B (zh) 2020-04-09 2020-04-09 一种用于纳米孔测序建库的位置锚定条码系统
CN202010276679.2 2020-04-09

Publications (1)

Publication Number Publication Date
WO2021203461A1 true WO2021203461A1 (fr) 2021-10-14

Family

ID=71651430

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/085645 WO2021203461A1 (fr) 2020-04-09 2020-04-20 Système de code à barres d'ancrage de position pour construction de banque de séquençage de nanopores

Country Status (2)

Country Link
CN (1) CN111440846B (fr)
WO (1) WO2021203461A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112029823B (zh) * 2020-09-03 2021-07-23 江苏先声医疗器械有限公司 纳米孔测序平台的宏基因组建库方法及其试剂盒
CN112176032B (zh) * 2020-10-16 2021-10-26 广州市达瑞生物技术股份有限公司 一个呼吸道病原体的纳米孔测序建库的引物组合及其应用
CN114480740B (zh) * 2022-02-18 2023-10-24 杭州柏熠科技有限公司 适用于15种植物检疫病毒的靶向测序建库及检测方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105986324A (zh) * 2015-02-11 2016-10-05 深圳华大基因研究院 环状小rna文库构建方法及其应用
CN106282161A (zh) * 2016-08-12 2017-01-04 成都诺恩生物科技有限公司 特异捕获并重复复制低频率dna碱基变异的方法及应用
CN110475864A (zh) * 2017-02-02 2019-11-19 纽约基因组研究中心公司 用于识别或量化在生物样品中的靶标的方法和组合物
WO2020036926A1 (fr) * 2018-08-17 2020-02-20 Cellecta, Inc. Préparation multiplex de fragments d'adn spécifiques à un gène à code à barres

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2862552A1 (fr) * 2012-01-26 2013-08-01 Nugen Technologies, Inc. Compositions et procedes pour l'enrichissement en sequence d'acide nucleique ciblee et la generation d'une banque a efficacite elevee
EP3143159B1 (fr) * 2014-05-13 2019-01-02 Life Technologies Corporation Systèmes et procédés pour la validation de résultats de séquençage
CN105989249B (zh) * 2014-09-26 2019-03-15 南京无尽生物科技有限公司 用于组装基因组序列的方法、系统及装置
GB201616590D0 (en) * 2016-09-29 2016-11-16 Oxford Nanopore Technologies Limited Method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105986324A (zh) * 2015-02-11 2016-10-05 深圳华大基因研究院 环状小rna文库构建方法及其应用
CN106282161A (zh) * 2016-08-12 2017-01-04 成都诺恩生物科技有限公司 特异捕获并重复复制低频率dna碱基变异的方法及应用
CN110475864A (zh) * 2017-02-02 2019-11-19 纽约基因组研究中心公司 用于识别或量化在生物样品中的靶标的方法和组合物
WO2020036926A1 (fr) * 2018-08-17 2020-02-20 Cellecta, Inc. Préparation multiplex de fragments d'adn spécifiques à un gène à code à barres

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANNA L. MCNAUGHTON, HANNAH E. ROBERTS, DAVID BONSALL, MARIATERESA DE CESARE, JOLYNNE MOKAYA, SHEILA F. LUMLEY, TANYA GOLUBCHIK, PA: "Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)", SCIENTIFIC REPORTS, vol. 9, 7081, 1 December 2019 (2019-12-01), pages 1 - 14, XP055856580 *
BRANDON D. WILSON, MICHAEL EISENSTEIN, H. TOM SOH: "High-Fidelity Nanopore Sequencing of Ultra-Short DNA Targets", ANALYTICAL CHEMISTRY, vol. 91, no. 10, 21 May 2019 (2019-05-21), US, pages 6783 - 6789, XP055856583, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.9b00856 *
ZHANG DE-FANG, MA QIU-YUE, YIN TONG-MING, XIA TAO: "The Third Generation Sequencing Technology and Its Application", CHINA BIOTECHNOLOGY, vol. 33, no. 5, 31 December 2013 (2013-12-31), pages 125 - 131, XP055856586, DOI: 10.13523/j.cb.20130520 *

Also Published As

Publication number Publication date
CN111440846A (zh) 2020-07-24
CN111440846B (zh) 2020-12-18

Similar Documents

Publication Publication Date Title
WO2021203461A1 (fr) Système de code à barres d'ancrage de position pour construction de banque de séquençage de nanopores
CN108893466B (zh) 测序接头、测序接头组和超低频突变的检测方法
CN107002292B (zh) 一种核酸的双接头单链环状文库的构建方法和试剂
CN109971827B (zh) 血浆dna的建库方法和建库试剂盒
CN106048009B (zh) 一种用于超低频基因突变检测的标签接头及其应用
CN111748551B (zh) 封闭序列、捕获试剂盒、文库杂交捕获方法及建库方法
WO2013142389A1 (fr) Méthodes permettant de faire baisser le taux d'erreur observé lors d'un séquençage adn massivement parallèle en faisant appel à un séquençage consensus duplex
CN111073961A (zh) 一种基因稀有突变的高通量检测方法
CN108517567B (zh) 用于cfDNA建库的接头、引物组、试剂盒和建库方法
WO2021227129A1 (fr) Adaptateur universel de séquençage à haut débit et application associée
CN111154916B (zh) 呼吸道病原多重rpa检测用引物群、检测试剂及试剂盒
CN110734967B (zh) 一种接头组合物及其应用
WO2018108328A1 (fr) Procédé pour augmenter le débit d'un séquençage de molécule unique par concaténation de fragments d'adn court
WO2023221308A1 (fr) Procédé de capture hybride en phase liquide et kit de test associé
Cheng et al. Methods to improve the accuracy of next-generation sequencing
WO2019001187A1 (fr) Amorce de détection de puce à adn de phase multi liquide, kit et méthode pour distinguer rapidement cinq pathogènes dans des voies respiratoires de souris
WO2021253372A1 (fr) Construction de bibliothèque sans pcr à haute compatibilité et procédé de séquençage
CN108359723B (zh) 一种降低深度测序错误的方法
JP2024504198A (ja) ヒトマイクロサテライトの不安定性を検出するためのバイオマーカー群及びその応用
WO2019227331A1 (fr) Procédé de construction d'une bibliothèque de séquences de région variable, procédé de séquençage et kit associé
CN108728515A (zh) 一种使用duplex方法检测ctDNA低频突变的文库构建和测序数据的分析方法
WO2016119448A2 (fr) Molécule de référence exogène artificielle pour comparer des types et l'abondance naturelle entre des micro-organismes d'espèces et de genres différents
WO2023221307A1 (fr) Sonde pour l'enrichissement cible d'acide nucléique
WO2016109981A1 (fr) Procédé de détection à haut débit de produit de synthèse d'adn
CN111926394B (zh) 基于宏基因组学的建库方法和检测试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20930290

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20930290

Country of ref document: EP

Kind code of ref document: A1