WO2012083845A1 - Procédés pour le retrait de fragments de vecteur dans une banque de séquençage et leur utilisation - Google Patents

Procédés pour le retrait de fragments de vecteur dans une banque de séquençage et leur utilisation Download PDF

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WO2012083845A1
WO2012083845A1 PCT/CN2011/084303 CN2011084303W WO2012083845A1 WO 2012083845 A1 WO2012083845 A1 WO 2012083845A1 CN 2011084303 W CN2011084303 W CN 2011084303W WO 2012083845 A1 WO2012083845 A1 WO 2012083845A1
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library
sequencing
sequencing library
probe
vector
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PCT/CN2011/084303
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English (en)
Chinese (zh)
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胡帅星
武靖华
樊帆
陈琳
赵美茹
王俊
汪建
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深圳华大基因科技有限公司
深圳华大基因研究院
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Publication of WO2012083845A1 publication Critical patent/WO2012083845A1/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

Definitions

  • the present invention relates to the field of molecular biology, particularly in the field of genome sequencing, and in particular, to methods for removing carrier fragments in sequencing libraries and uses thereof. More specifically, the present invention provides a method for removing a vector fragment in a sequencing library, a method for sequencing a genomic clone library, and a kit for genome sequencing. Background technique
  • De nove sequencing is also called de novo sequencing. It can sequence the genome of a certain species without any gene sequence information. After sequencing, bioinformatics analysis method can be used to splicing and assembling the sequences obtained by sequencing. A genomic sequence map of the species is obtained, which can be applied to other genomics, genetic studies of the species. Therefore, the e wove sequencing of biological organisms is of great significance.
  • Illumina's DNA sequencing platform specifically, by preparing a sequencing library of the organism and then sequencing it.
  • the sequencing library preparation method based on Illumina's DNA sequencing platform is divided into small-sequence DNA sequencing library preparation and large-segment DNA sequencing library preparation.
  • Fosmid clone library as a template (also sometimes referred to as construction in this paper).
  • the Fosmid clone library is obtained by transferring the total DNA of a certain organism together with the Fosmid vector into a host cell in a recombinant form, and then forming a plurality of clones by cell proliferation.
  • a suitable length of the insertable genomic DNA fragment is about 40 kb.
  • Construction of a Fosmid small fragment sequencing library refers to the preparation of a sequencing library by mixing a certain number of Fosmid clones together as a library template.
  • the Fosmid vector was also disrupted into a small number of small fragments of DNA below lkb due to disruption of the Fosmid clone during the database construction process. Therefore, the sequencing library contains a large number of Fosmid vector fragments. Therefore, when the entire sequencing library is sequenced, a large amount of unnecessary vector sequence data is generated, and the unnecessary sequence data also affects subsequent data analysis, resulting in Sequencing resources are wasted, sequencing costs are high, and efficiency is low.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the present invention provides methods for removing vector fragments in a sequencing library and uses thereof, so that the Fosmid vector fragment will be removed prior to sequencing the sequencing library using a Solexa DNA sequencer to avoid sequencing the Fosmid vector. Reduce unnecessary data reading and analysis, which can significantly reduce the cost of sequencing and improve sequencing efficiency.
  • the invention provides a method for removing a vector fragment in a sequencing library.
  • the method comprises the steps of:
  • a labeled probe is provided that is capable of hybridizing to a vector fragment.
  • the vector is a Fosmid vector.
  • hybridization refers to two single-stranded nucleic acid molecules having complementary sequences to each other under certain conditions (suitable temperature and ionic strength, etc.) annealed to form a double strand according to the principle of complementary pairing of bases.
  • the process of nucleic acids also known as nucleic acid hybridization.
  • the nucleic acid hybridization can be carried out between Li A-Li A or between Li A-RNA or RNA-RNA, and base pairing can be performed as long as there is a complementary sequence in both of the hybrids.
  • nucleic acid hybridization includes solid-liquid phase hybridization and liquid phase hybridization, wherein liquid phase hybridization is a hybridization reaction carried out in a solution, which means that a nucleic acid molecule to be tested is hybridized with a known nucleic acid molecule (probe) in a solution to form a hybrid. Complex.
  • vector fragment may be the entire Fosmid vector, or a fragment of all vectors formed by disrupting the entire Fosmid vector, whereby the expression “probe” and vector Fragmentation of the fragment "represents that the sequence of the probe can be complementary to the entire Fosmid vector sequence, thereby enabling hybridization with the entire Fosmid vector, or the sequence of the probe can be complementary to the sequence of the fragments of all of the above vectors, thereby enabling fragmentation with all vectors. Hybridization is carried out.
  • the probe is hybridized to the sequencing library such that the probe and the vector fragment form a labeled double-stranded nucleic acid.
  • the hybridized duplex can be isolated and detected using the label on the probe, wherein the label is known in the art and can include, but is not limited to, Radioactive co-location Or a nuclides, biotin, acridinium ester or polyA.
  • the label is known in the art and can include, but is not limited to, Radioactive co-location Or a nuclides, biotin, acridinium ester or polyA.
  • corresponding nucleic acid isolation and detection methods known in the art can be utilized, including but not limited to hydroxyapatite (HAP) or affinity adsorption (see, for example, Henegariu 0 et al. (1999).
  • HAP hydroxyapatite
  • affinity adsorption see, for example, Henegariu 0 et al. (1999).
  • PCR 3 PCR in situ hybridization: a practical approach, Volume 3. Oxford: Oxford University Press, which is incorporated herein by reference in its entirety, the hybridization product is isolated and tested.
  • the expression "probe and vector fragment forms a labeled double-stranded nucleic acid” means that the probe can hybridize with the entire Fosmid vector to form a labeled double-stranded nucleic acid, or it can be a probe and all of the above vectors.
  • the fragment hybridizes to the labeled double-stranded nucleic acid.
  • the probe is subjected to a liquid phase hybridization reaction with a sequencing library.
  • the probe can be hybridized with the sequencing library in a mass ratio of from about 1:1 to about 2:1.
  • it may further comprise adding a linker blocking agent to the sequencing library.
  • the ratio of linker blocking agent to sequencing library may range from about 0.3 pM/ng to 0.8 pM/ng. According to a specific example of the invention, the ratio of the linker blocking agent to the sequencing library is 0.5 pM/ng. According to an embodiment of the present invention, the hybridization reaction may take a time of 1, 4, 16, 24 or more hours.
  • the labeled double-stranded nucleic acid is then removed using the labeled molecular entity on the specific binding probe to remove the vector fragment in the sequencing library.
  • the aforementioned labeled probe may be a biotinylated probe and the molecular entity is avidin.
  • the molecular entity is streptavidin, and according to a specific example of the invention, the molecular entity is formed on the magnetic beads.
  • the inventors have surprisingly found that the method for removing vector fragments in a sequencing library according to an embodiment of the present invention enables efficient removal of vector fragments in a Fosmid small fragment sequencing library for sequencing based on a high throughput sequencing platform such as Illumina Solexa
  • the platform after sequencing the processed sequencing library, can effectively avoid waste of sequencing resources and generate unnecessary sequencing data, ultimately reducing the cost of sequencing and improving the sequencing efficiency.
  • the method for removing a vector fragment in a sequencing library of the present invention may comprise the following steps:
  • the vector is used to construct a genomic clone library, and is preferably a Fosmid vector.
  • the probe is labeled with biotin and the molecular entity that specifically binds to the label is avidin.
  • the molecular entity is streptavidin, and according to one embodiment of the present invention, streptavidin may be formed on the magnetic beads.
  • the probe is subjected to a liquid phase hybridization reaction with a sequencing library.
  • the biotinylated probe can be prepared by the following steps:
  • the PCR product can be fragmented using a Covaris instrument.
  • the PCR product is specifically fragmented into fragments of about 300 bp according to the present invention.
  • the hybridization reaction can be carried out by denaturation of the probe and the sequencing library at 95 °C, followed by annealing at 65 °C.
  • the probe and the sequencing library can be subjected to a hybridization reaction at a mass ratio of about 1:1 to about 2:1.
  • a linker blocker can optionally be added to the sequencing library prior to hybridization of the probe to the sequencing library.
  • the linker blocking agent is a nucleic acid capable of hybridizing to a linker used in the construction of the sequencing library, and preferably, the nucleotide sequence of the linker blocker is completely complementary to the nucleotide sequence of the linker, thereby When the probe is hybridized to the sequencing library, the linker is hybridized to the linker to avoid hybridization of the linker to the probe and pairing between the linkers.
  • the ratio of the linker blocking agent to the library may be from about 0.3 pM/ng to about 0.8 pM/ng, for example 0.5 pM/ng.
  • the amount of the linker blocking agent added is too large, the removal efficiency of the vector fragment in the library is lowered.
  • the amount of the linker blocking agent added is too small, a large amount of non-vector library DNA fragments are captured by the probe. Remove.
  • the hybridization can be carried out for 1, 4, 16, 24 or more hours.
  • the present invention provides a method for sequencing a genomic clone library, comprising the steps of:
  • a sequencing library is constructed using a genomic clone library in which a vector fragment is included in the sequencing library.
  • the genomic clone library is a Fosmid clone library and the vector is a Fosmid vector.
  • the sequencing library can be constructed by: fragmenting the DNA in the genomic clone library to obtain a DNA fragment; ligating the linker to both ends of the DNA fragment to obtain a ligation product; and PCR-amplifying the ligation product , in order to obtain an amplification product; and recovering and purifying the amplification product, the amplification product constituting a sequencing library.
  • the vector fragment in the sequencing library is removed using a method for removing a vector fragment in a sequencing library according to an embodiment of the present invention.
  • the vector fragment in the sequencing library may be removed after the sequencing library is constructed, or during the process of constructing the sequencing library; according to a specific example of the present invention, after the DNA in the genomic clone library is fragmented , removing the vector fragment in the sequencing library.
  • sequencing library after removal of the vector fragment was sequenced.
  • the method of sequencing is not particularly limited. According to one embodiment of the invention, sequencing can be performed using a Solexa sequencer.
  • the method for sequencing a genomic clone library according to an embodiment of the present invention can efficiently sequence a genomic clone library, that is, a Fosmid small fragment sequencing library, since the method can effectively remove Fosmid small fragments.
  • a genomic clone library that is, a Fosmid small fragment sequencing library
  • the method can be effectively applied to the centralized commercial multi-species genome sequencing which is carried out on a large scale at present, thereby saving cost and improving efficiency, and further, capable of performing genomic clone library according to an embodiment of the present invention.
  • the method of sequencing is applied to new areas such as analysis, disease diagnosis, and personalized (individualized) medicine.
  • the method for sequencing a genomic clone library of the present invention may comprise the following steps:
  • the genomic clone library is a Fosmid clone library and the vector is a Fosmid vector.
  • a method for removing a vector fragment in a sequencing library according to the present invention can be utilized to remove a vector fragment in a sequencing library.
  • the construction of the sequencing library can be performed by the following steps:
  • the vector fragment in the sequencing library can be removed after construction of the sequencing library. Root According to some embodiments of the invention, the vector fragments in the sequencing library can be removed during the construction of the sequencing library. According to a specific example of the present invention, the vector fragment can be removed after fragmenting the DNA in the genomic library.
  • the sequencing library can be sequenced using a Solexa sequencer.
  • the present invention provides a kit for genome sequencing, comprising: a vector for constructing a genomic clone library; a labeled probe capable of interacting with the vector or The fragment is hybridized; and a molecular entity capable of specifically binding to the label on the probe.
  • the vector is a Fosmid vector.
  • the probe is a biotinylated probe and the molecular entity is avidin.
  • the molecular entity is streptavidin.
  • the molecular entity is streptavidin formed on the magnetic beads.
  • kits of the invention may also include other reagents required for genomic clone library construction, sequencing, etc., which may further include linkers and linker blocking agents in accordance with embodiments of the present invention.
  • linkers and linker blocking agents in accordance with embodiments of the present invention.
  • a kit according to an embodiment of the present invention can be effectively used for genome sequencing, and in particular, a vector in a kit can be used to efficiently construct a library of genomic clones, thereby utilizing labeled probes and molecules in the kit.
  • the entity is capable of efficiently removing the vector fragments in the library, thereby enabling efficient genome sequencing of the library from which the vector fragments are removed based on a high throughput sequencing platform.
  • the application of the kit of the present invention to genome sequencing can effectively avoid waste of sequencing resources and generate unnecessary sequencing data, thereby reducing the cost of sequencing, improving the sequencing efficiency, and obtaining accurate sequencing results and easy data analysis.
  • the kit for genome sequencing of the present invention may comprise: a vector for constructing a genomic clone library, a labeled probe capable of hybridizing with a vector or a fragment thereof, and capable of specifically binding The labeled molecular entity of the probe.
  • the vector is a Fosmid vector.
  • the probe is labeled with biotin and the molecular entity that specifically binds to the label is avidin, and according to a specific example of the invention, the preferred molecular entity is streptavidin.
  • streptavidin may be formed on the magnetic beads.
  • the kit may also include other reagents including, but not limited to, linkers and linker blocking agents.
  • the probe was prepared by a PCR reaction using a Fosmid vector (the sequence of which is shown in SEQ ID NO: 1) as a template.
  • the reaction system of PCR is as follows:
  • the dNTP mixed solution used is a mixed solution of biotin-dNTP and normal dNTP in a ratio of 15:85, and the final concentration is 1 ⁇ .
  • the forward primer for preparing the probe was: 5'-CCTGGGGTGCCTAATGAGTG-3' (SEQ ID NO: 2).
  • Reverse primer for the preparation of the probe was: 5'-CGTCGTTTTACAACGTCGTGA-3' (SEQ ID NO: 3).
  • the PCR reaction conditions were: 95 ° C, 2 minutes; 12 cycles of 95 ° C, 30 seconds, 65 ° C, 30 seconds, 72 ° C, 8 minutes; 72 ° C, 10 minutes; 4 ° C storage.
  • the PCR product was purified using the DNA clean and ConcentratorTM-25 kit, and then the PCR product was disrupted using a Covaris S2 instrument to obtain a DNA fragment, which was then determined by gel electrophoresis to determine the size of the DNA fragment.
  • the test results showed that the size of the DNA fragment was mainly about 300 bp.
  • the DNA fragment was purified by MinElute PCR Purification Kit and dissolved in 20 ⁇ l elution buffer to obtain a biotin-labeled probe. Then, use Qubit (HS) to quantify the concentration of the probe and set aside.
  • HS Qubit
  • the Fosmid clone library of Puccinia striiformis f.sp 'rid genomic DNA was prepared using the CopyControlTM HTP Fosmid Library Construction Kit (Epicentre, USA) according to the manufacturer's detailed instructions.
  • SC hybridization buffer and SC hybridization component A are derived from the sequence capture hybridization kit.
  • the amount of the library was 120 ng
  • the amount of the probe was 120 ng (quantified by Qubit)
  • a linker blocking agent was added to the hybridization system.
  • the linker blocker was obtained from the tag sequencing primer kit (Illumina) and the mixed Rd2 sequencing primer (Multixing Rd2 Sequencing Primer kit, Illumina), wherein the sequence of the linker blocker is as follows:
  • Linker - sputum 1 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCC TACACG ACGCTCTTCCGATCT-3' (SEQ ID NO: 4);
  • Linker - sputum 2 5'-ACAAGCAGAAGACGGCATACGAGATAAGCAATGGTGA CTGGA GTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 5).
  • the amount of both linker blocking agents was 0.06 nM, and the ratio of the amount of the linker to the library was 0.5 pM/ng.
  • hybridization conditions are: denaturation at 95 ° C 10
  • hybridization was carried out at 65 ° C according to the time specified in the table below to obtain a hybridization product, which was used.
  • Library DNA (i.e., vector fragment) capable of hybridizing with the probe in the above hybridization product was captured and isolated using streptavidin magnetic beads (M280 magnetic beads) according to the manufacturer's instructions, and then derived from samples 1 and 2
  • streptavidin magnetic beads M280 magnetic beads
  • library DNA i.e., vector fragments hybridized with the probes from samples 1 and 2, respectively, were collected and labeled 24-1-V and 4- 12-V.
  • the library DNAs obtained above namely 24-l-D, 4- 12-D, 24-1-V and 4- 12-V, were purified by QIAquick PCR Purification Kit according to the manufacturer's instructions.
  • the library DNA obtained above was subjected to PCR amplification using PFX enzyme, wherein the PCR reaction system was as follows:
  • the forward primer for library DNA amplification is: 5'-CAAGCAGAAGACGGCATACGA -3' (SEQ ID NO: 6)
  • reverse primer for library DNA amplification 5'- AATGATACGGCGACCACCGAGATC -3' (SEQ ID NO: 7).
  • the PCR reaction conditions were: 94 ° C, 2 minutes; 12 cycles of 94 ° C, 15 seconds, 58 ° C, 30 seconds, 72 ° C, 30 seconds; 72 ° C, 5 minutes; 4 ° C storage. Thereby, an amplification product was obtained.
  • the amplified product was purified, dissolved in 30 ⁇ l of ultrapure water, and the concentration of the obtained amplification product was measured by Nanodrop, and the results were as follows:
  • the effect of removing the vector fragment in the library by the method for removing the vector fragment in the sequencing library of the present invention is analyzed by Q-PCR.
  • a sample which was not treated by the method of the present invention was selected as a control.
  • the untreated sample and the treated sample were diluted to the same concentration, and Q-PCR was performed to detect the content of the vector fragment in the two samples.
  • the degree of enrichment indicates a multiple of the amount of the carrier fragment in the control sample relative to the amount of the carrier fragment in the treated sample, and therefore, the effect of carrier removal can be obtained from the value of the enrichment degree, for example, when the enrichment degree is N , which indicates that the sample treated by the method of the present invention removes the 1-1/N carrier fragment.
  • the library DNA from which the vector fragment was removed i.e., the above 24-1-1D and 4112-D
  • the library DNA from which the vector fragment was not removed i.e., the sequencing library of the wheat stripe rust DNA
  • the Q-PCR reaction system is as follows:
  • SYBR Premix and Rox Reference Dye II are from SYBR@ Premix Ex TaqTM Among them, the forward primer for Q-PCR is: 5'-TTgTTCCCACgCCTGCTGAgTTGT-3' (SEQ ID NO: 8), and the reverse primer for Q-PCR is: 5'-ATCCCgAATTTgCTCCTCCATCCAC-3' (SEQ ID NO : 9 Q-PCR reaction conditions are: 95 ° C, 30 seconds; 40 cycles of 95 ° C, 15 seconds, 60 ° C, 1 minute.
  • the degree of enrichment of the vector fragment is greater than 5, which indicates that the content of the vector fragment in the sample treated by the method of the present invention is less than 1/5 of that of the control sample, thereby indicating that the method of the present invention removes the initial library. At least 80% of the vector fragments.
  • the probe prepared by the method for preparing a probe used in Example 1 was used.
  • a Fosmid clone library was constructed using blood genomic DNA from a Chinese adult male, and then a multi-sample preparation oligonucleotide kit (Illumina, PE-400-) was used according to the manufacturer's instructions. 1002) A sequencing library was constructed, called a YH Fosmid sequencing library. Then, the probe prepared above and the YH Fosmid sequencing library (hereinafter sometimes referred to simply as "library” in the present example) were prepared and hybridized according to the ratios in the following table:
  • Joint sealer 2 0.025 nM
  • the water was made up to 15 ⁇ wherein, in the above hybrid system, the mass ratio of the library to the probe was 1:2, and the linker blocking agent was the same as the linker of Example 1.
  • the hybridization conditions were: denaturation at 95 ° C for 10 minutes, followed by hybridization at 65 ° C for 24 hours, thereby obtaining a hybridization product, which was used.
  • the library DNA capable of hybridizing with the probe that is, the vector fragment
  • the library DNA from which the vector fragment is removed is collected. It was labeled as YH-DNA-1 and its concentration was determined. The results are as follows:
  • the library DNA (i.e., vector fragment) that hybridizes to the probe is collected and labeled as YH-Vector-1.
  • the obtained library DNA was purified using QIAquick PCR Purification Kit according to the manufacturer's instructions and used.
  • the method of removing the vector fragment in the library in this example was analyzed by Q-PCR.
  • the effect of Q-PCR is as follows:
  • the degree of enrichment of the vector fragment is greater than 5, indicating the sample treated by the method of the present invention.
  • the content of the carrier fragment was less than 1/5 of the control sample.
  • the method of the invention removes at least 80% of the vector fragments in the original library.
  • the method for removing a vector fragment in a sequencing library of the present invention, a method for sequencing a genomic clone library, and a kit for genome sequencing can effectively remove a vector fragment in a Fosmid small fragment sequencing library, thereby enabling It can be applied to the sequencing of genomic clone libraries, which can reduce the cost of sequencing and improve the efficiency of sequencing and data analysis.

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Abstract

La présente invention concerne des procédés pour le retrait de fragments de vecteur dans une banque de séquençage, des procédés pour banque de clones du séquençage de génomes et des trousses pour le séquençage des génomes. Les procédés pour le retrait de fragments de vecteur dans une banque de séquençage comprennent: la mise à disposition d'amorces étiquetées dans la banque de séquençage qui sont capables de s'hybrider avec des fragments de vecteur ; l'hybridation des amorces avec la banque de séquençage, de sorte que les amorces et les fragments de vecteur forment un acide nucléique double brin étiqueté ; et l'utilisation d'entité moléculaire étiquetée sur des amorces de liaison spécifique pour le retrait d'acide nucléique double brin et donc le retrait de fragments de vecteur dans la banque de séquençage.
PCT/CN2011/084303 2010-12-22 2011-12-20 Procédés pour le retrait de fragments de vecteur dans une banque de séquençage et leur utilisation WO2012083845A1 (fr)

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CN201010600214.4A CN102559856B (zh) 2010-12-22 2010-12-22 去除测序文库中的载体片段的方法
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CN102855398B (zh) * 2012-08-28 2016-03-02 中国科学院自动化研究所 基于多源信息融合的疾病潜在关联基因的获取方法
CN106029899B (zh) * 2013-09-30 2021-08-03 深圳华大基因股份有限公司 确定染色体预定区域中snp信息的方法、系统和计算机可读介质
ITUA20162640A1 (it) * 2016-04-15 2017-10-15 Menarini Silicon Biosystems Spa Metodo e kit per la generazione di librerie di dna per sequenziamento massivo parallelo
EP3887540B1 (fr) * 2018-11-30 2023-08-23 Illumina, Inc. Analyse d'analytes multiples à l'aide d'un seul dosage

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KIM ET AL.: "Cloning, Sequencing, and Characterization of the Pradimicin Biosynthetic Gene Cluster of Actinomadura hibisca P157-2", J. MICROBIOL. BIOTECHNOL, vol. 17, no. 5, 2007, pages 830 - 839 *

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