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WO2012037877A1 - Marqueurs d'adn et leurs utilisations - Google Patents

Marqueurs d'adn et leurs utilisations Download PDF

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Publication number
WO2012037877A1
WO2012037877A1 PCT/CN2011/079899 CN2011079899W WO2012037877A1 WO 2012037877 A1 WO2012037877 A1 WO 2012037877A1 CN 2011079899 W CN2011079899 W CN 2011079899W WO 2012037877 A1 WO2012037877 A1 WO 2012037877A1
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WIPO (PCT)
Prior art keywords
dna
pcr
index
free
tag
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PCT/CN2011/079899
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English (en)
Chinese (zh)
Inventor
章文蔚
田方
陈海燕
于竞
龚梅花
张艳艳
周妍
汪建
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Publication of WO2012037877A1 publication Critical patent/WO2012037877A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • 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
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support

Definitions

  • the invention relates to the field of nucleic acid sequencing technology, in particular to the field of DNA sequencing technology.
  • the invention relates to DNA tags for DNA sequencing and their use. More specifically, the present invention provides a DNA tag, an oligonucleotide, a DNA tag library, a preparation method thereof, a method for determining DNA sequence information, a method for determining sequence information of a plurality of DNA samples, and a method for constructing a DNA tag library.
  • a kit for constructing a DNA tag library Background technique
  • DNA sequencing technology is one of the important molecular biological analysis methods. It not only provides important data for basic biological research such as gene expression and gene regulation, but also plays an important role in applied research such as disease diagnosis and gene therapy. .
  • Solexa DNA Sequencing Platform Illumina
  • SBS Sequencing By Synthesis
  • the required sample volume high throughput, high accuracy, easy-to-operate automation platform and powerful functions, etc.
  • Paired-End sequencing User Guide Illumina part #1003880; Preparing samples for ChIP sequencing for DNA; Illumina part#l 1257047 Rev. A; mRNA sequencing sample preparation guide; Illumina part#1004898 Rev.D; Preparing 2- 5 kb samples for mate pair library sequencing; Illumina part #1005363 Rev.B, which is incorporated herein by reference in its entirety.
  • Illumina has introduced a DNA tag (also known as index) database building method based on the Solexa DNA sequencing platform. As shown in Fig. 1, in the DNA tag construction process, three PCR primers were used, and a DNA tag library was constructed by PCR. (Preparing samples for multiplexed paired-End sequencing; Illumina part#1005361 Rev.B, by reference Incorporate it in its entirety).
  • the inventors of the present application found that the above-described method for preparing a tag library has some drawbacks: First, Illumina currently only provides 12 tag sequences of 6 bp in length, and the number of tags is small, and as the Solexa sequencing throughput increases, It is impossible to mix and sequence a large number of samples, which will waste the sequencing resources and affect the sequencing flux. Second, the above label construction method is to introduce the tag sequence into the library of the target fragment by PCR reaction, and the PCR amplification of the target fragment The amplification process requires the use of three PCR primers (two common PCR primers and one PCR tag primer, as shown in Figure 1), time-consuming consumables, and inefficient PCR amplification.
  • the linker used in the above label construction method does not include a tag sequence. Therefore, when a plurality of sample DNAs are sequenced, the tag libraries of each sample need to be independently constructed, that is, the tag sequences are respectively introduced by PCR reaction. Then, each label library is separately cut and recovered, and then the respective label libraries obtained by the gel extraction are mixed, and finally the mixture of the plurality of sample label libraries can be sequenced, which is time-consuming and laborious, and high in cost.
  • a DNA tag (herein sometimes referred to as a "tag") that can be used to construct a library of DNA tags is proposed.
  • the invention proposes a set of isolated DNA tags.
  • the sample source of the DNA can be accurately characterized by linking the DNA tag to the sample DNA or its equivalent.
  • a DNA tag library of a plurality of samples (herein, sometimes referred to as a "tag library”;) can be simultaneously constructed, thereby allowing sequencing by mixing DNA tag libraries derived from different samples.
  • the library is sequenced to increase the sequencing efficiency and throughput of the DNA tag library.
  • the inventors have surprisingly found that the construction of a DNA tag library using a DNA tag according to an embodiment of the present invention enables precise discrimination of a plurality of DNA tag libraries, and the resulting sequencing data results are very stable and reproducible.
  • the invention also provides a set of isolated oligonucleotides for introducing the above DNA tag into a sample DNA or an equivalent thereof.
  • these oligonucleotides (also referred to herein as "DNA PCR-Free tag linkers", “PCR-Free tag linkers”) have respectively implemented according to the invention as described above
  • the DNA tag of the example has a sticky end T, and thus, the corresponding DNA tag can be introduced into the DNA or its equivalent by a ligation reaction.
  • the sequence is shown in Table 1 below (the sequence directions shown in the table are all 5' - 3' directions).
  • a DNA PCR-Free tag linker having a Y-form structure can be formed by subjecting DNA PCR-Free linker 1.0 to PCR-Free Index-N in an equimolar annealing treatment.
  • DNA tag sequence (DNA Index-N) and its corresponding DNA PCR-Free tag linker sequence
  • a DNA tag can be efficiently introduced into the DNA of the sample or its equivalent, whereby a DNA tag library having a DNA tag can be constructed.
  • the inventors have surprisingly found that when constructing a DNA tag library containing various DNA tags with oligonucleotides having different tags for the same sample, the stability and reproducibility of the resulting sequencing data results are very it is good.
  • the human whole blood sample DNA tag library constructed using DNA Indexl-161 exhibits a correlation of at least 0.99 when data analysis is performed using the pearson coefficient. Details of the specific algorithm for the pearson coefficient can be found in the relevant literature, for example: t Hoen, PA, Y.
  • the present invention provides a method of preparing a DNA tag library. According to an embodiment of the present invention, comprising: providing a DNA template having two oligonucleotide strands; adding a base A at each of the two oligonucleotide strands of the DNA template; Connecting a linker comprising one selected from the above-described group of isolated DNA tags according to an embodiment of the present invention to each other at both ends of the DN A template to obtain a ligation product; and separately recovering the ligation product, the ligation product
  • the DNA tag library is constructed.
  • a DNA tag according to an embodiment of the present invention can be efficiently introduced into a DNA tag library constructed for sample DNA.
  • the DNA tag library can be sequenced to obtain sequence information of the sample DNA and information on the DNA tag, thereby distinguishing the source of the sample DNA.
  • the inventors have surprisingly found that when the same sample is used, based on the above method, when a DNA tag library containing various DNA tags is constructed using oligonucleotides having different tags, the stability of the obtained sequencing data results is Repeatability is very good.
  • the present invention also provides a DNA tag library obtained by the method of constructing a DNA tag library according to an embodiment of the present invention.
  • the present invention also provides a method of determining DNA sample sequence information.
  • a method of determining DNA sample sequence information comprising: constructing a DNA tag library of the DNA sample according to a method of constructing a DNA tag library according to an embodiment of the present invention; and sequencing the DNA tag library to determine a sequence of the DNA sample information.
  • the sequence information of the DNA sample in the DNA tag library and the sequence information of the DNA tag can be efficiently obtained, thereby enabling differentiation of the source of the DNA sample.
  • the inventors have surprisingly found that the use of the method according to an embodiment of the present invention to determine DNA sample sequence information can effectively reduce the problem of data production bias and can accurately distinguish a plurality of DNA tag libraries.
  • the present invention also provides a method of determining sequence information of a plurality of DNA samples.
  • the method comprises the steps of: establishing, for each of the plurality of samples, a DNA tag library of the DNA sample independently of the method of constructing a DNA tag library according to an embodiment of the present invention, wherein Different DNA samples are labeled with DNA tags of different and known sequences, wherein the plurality of samples are 2-161 Generating a DNA tag library of the plurality of samples to obtain a DNA tag library mixture; sequencing the DNA tag library mixture using Solexa sequencing technology to obtain sequence information of the DNA sample and the tag Sequence information; and classifying sequence information of the DNA sample based on sequence information of the tag to determine DNA sequence information of the plurality of samples.
  • the method according to an embodiment of the present invention can make full use of high-throughput sequencing technology, for example, using Solexa sequencing technology, and simultaneously sequencing DNA tag libraries of various samples, thereby improving the efficiency and sequencing of DNA tag library sequencing.
  • the amount, at the same time, can improve the efficiency of determining the sequence information of a variety of DNA samples.
  • a kit for constructing a DNA tag library comprising: 161 isolated oligonucleotides, said isolated oligonucleotide, according to an embodiment of the present invention
  • Figure 1 Schematic diagram showing the construction of a DNA tag library provided by Illumina
  • Figure 2 Schematic diagram showing a DNA tag library construction method according to an embodiment of the present invention
  • Figure 3 shows a construction according to an embodiment of the present invention Electrophoresis results of 44 DNA tag libraries.
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first”, “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, “multiple” means two or more unless otherwise stated.
  • the present invention proposes a number of isolated DNA tags.
  • SEQ ID NO: (2N-1) any integer of 1-161.
  • DNA as used in the present invention may be any polymer comprising deoxyribonucleotides including, but not limited to, modified or unmodified DNA.
  • a DNA tag according to an embodiment of the present invention, a DNA tag library having a tag is obtained by linking the DNA tag to the DNA of the sample or its equivalent, and the sequence of the sample DNA and the sequence of the tag can be obtained by sequencing the DNA tag library. Further, based on the sequence of the tag, the sample source of the DNA can be accurately characterized.
  • a DNA tag library of a plurality of samples can be simultaneously constructed, and the DNA sequence of the sample can be classified based on the DNA tag by mixing and simultaneously sequencing the DNA tag library derived from different samples.
  • DNA tag attached to the DNA of the sample or its equivalent should be understood in a broad sense, including that the DNA tag can be directly linked to the DNA of the sample to construct a DNA tag library, and can also be associated with the DNA of the sample. Nucleic acid of the same sequence (for example, can be the corresponding RNA The sequence or cDNA sequence, which has the same sequence as the DNA, is ligated.
  • the inventors of the present application found that: In the present invention, in order to design an effective DNA tag, it is first necessary to consider the problem of recognizability and recognition rate between tag sequences. Second, in the case of a label mix of less than 12 samples, the GT content of each base site on the mixed label must be considered. Because the excitation fluorescence of the bases G and T is the same in the Solexa sequencing process, the excitation lights of the bases A and C are the same, so the "balance" of the base “GT” content and the base “AC” content must be considered. The base base "GT” content is 50%, which guarantees the highest label recognition rate and the lowest error rate. Finally, consider the repeatability and accuracy of the data output.
  • a set of DNA tags In order to achieve efficient construction of the DNA tag library and sequencing, a set of DNA tags must be constructed to ensure reliable results and high reproducibility. The same DNA sample ensures that a library of DNA tags constructed using different tags in the set of DNA tags will result in consistent sequencing results, thus ensuring reliable and reproducible results. In addition, it is also necessary to avoid the appearance of 3 or more consecutive bases in the tag sequence, because 3 or more consecutive bases increase the error rate of the sequence during synthesis or sequencing, and also Try to avoid the DNA tag connector itself forming a hairpin structure.
  • the inventors of the present application performed a large number of screening work, and selected a set of isolated DNA tags according to an embodiment of the present invention, which are respectively represented by the nucleotides represented by SEQ ID NO: (2N-1)
  • the sequence is as shown in Table 1 above and will not be described again.
  • the inventors found that the differences between these tags are at least 4 bases, that is, at least 4 base sequences are different, and when any one of the 8 bases of the tag has a sequencing error or a synthetic error, Does not affect the final identification of the label.
  • These tags can be applied to the construction of any DNA tag library. There are no reports on the construction of these tags for DNA sample sequencing and sequencing by Solexa.
  • the DNA tag used is a nucleic acid sequence of 8 bp in length, and the difference between the tags is more than 4 bases, the set of DNA tags consisting of: 161 DNA At least 10 of the labels or DNA strands differing by 1 base, or at least 20, or at least 30, or at least 40, at least 50, or at least 60, or at least 70, or at least 80 , or 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or all 161.
  • the set of DNA tags preferably includes at least 161 DNA tags shown in Table 1 DNA Index 1 ⁇ DNA Index 10, or DNA Index 1 ⁇ DNA Index 20, or DNA Index 21 ⁇ DNA Index30 , or DNA Index31 - DNA Index40 , or DNA Index ⁇ DNA Index50 , or DNA Index51 ⁇ DNA Index60 , or DNA Index61 ⁇ DNA Index70 , or DNA Index71 ⁇ DNA Index80 , or DNA Index81 ⁇ DNA Index90 , or DNA Index91 ⁇ DNA IndexlOO, or DNA Index 101 - DNA Index 110, or DNA Index 111 - DNA Index 120, or DNA Index 121 ⁇ DNA Index 130, or DNA Index l ⁇ DNA Index 140, or DNA Indexl41 ⁇ DNA Index 150, or DNA Index 151 - DNA Indexl61, or a combination of any two or more of them.
  • the 1 base difference comprises a substitution, addition or deletion of 1 base in the sequence of 161 tags shown in Table 1.
  • the present invention also provides the use of a tag according to an embodiment of the present invention for the construction and sequencing of a DNA tag library, wherein the DNA tag linker of the DNA tag library comprises a DNA tag according to an embodiment of the present invention, thereby Each of the corresponding DNA tag adapters is constructed.
  • the DNA tag is inserted into a DNA PCR-Free tag linker, or ligated to the 3' end of the DNA linker with or without a linker, preferably into a DNA PCR-Free tag linker.
  • the linker is a 1-6 nucleotide sequence, preferably a 1-3 nucleotide sequence.
  • the invention provides a set of isolated oligonucleotides which can be used to introduce a DNA tag as described above into the DNA of a sample, thereby constructing a library of DNA tags.
  • the invention provides a set of isolated oligonucleotides, each of the set of isolated oligonucleotides having a sticky end T, and the isolated oligonucleotides having a first The chain and the second strand, the sticky end T, are formed on the first strand of each of the oligonucleotides.
  • the first strand is composed of the nucleotide represented by SEQ ID NO: 323
  • the corresponding oligonucleotides can be formed by annealing the first strand and the second strand constituting the corresponding oligonucleotide, respectively.
  • the above oligonucleotides respectively have the DNA tags according to the embodiments of the present invention as described above, and the oligonucleotides have sticky ends, and thus, the corresponding DNA tags can be linked by a ligation reaction. Introduced into the DNA of the sample or its equivalent. Specifically, the sequences of these oligonucleotides are as shown in Table 1 above, and are not described herein again.
  • oligonucleotide sequence DNA PCR-Free tag junction
  • Lasergene software http://www.dnastar.com/
  • the affinity parameter between the duplexes can be determined by analyzing the energy values formed between the two sequences, thereby predicting the most stable dimer overran formed by the DNA PCR-Free tag linker (the most stable dimer overran And the energy value, wherein the larger the absolute value of the energy value (kcal/mol), the more stable the result of the duplex is.
  • the following are the results of the above structural stability and affinity analysis of the 161 DNA PCR-Free tag linkers shown in Table 1 above. The results show that the "Y-type" structure formed by these DNA PCR-Free tag linkers is very stable.
  • the second structure of the DNA PCR-Free tag linker and the most stable dimer overall - "gamma type” structure and its energy value are provided below in accordance with an embodiment of the present invention.
  • the ost stable dimer overall: 12 bp , -22. B kcal /mol
  • the mo t stable dimer overall 12 bp, -22.8 kcsl/rrtoi
  • the mos stable diner overall ⁇ 2 hp f -22.8 kcsl/ino ⁇ GGGCA AGTAA 5 '
  • He mo t s able diraer overall 12 fo , -22. ⁇ kcal /mol
  • NCeede6 DA PRFr inxl2- p, / The most stahLe overall b kcaJ_mo.--- ?NCeede5 DA PRFr inxl2- ACACGTCT:AGTCACTGArA cr:GrAc ⁇ C TG;;r.,,
  • the mo t stable dimer overall 12 bp , -22 . 8 kcal /r o
  • the invention provides DNA PCR-Free tag junctions, these DNAs
  • the PCR-Free tag linker consists of a DNA PCR-Free linker 1.0 and a PCR-Free tag sequence, and these PCR-Free tag sequences include or consist of the following: 161 PCR-Free tag sequences shown in Table 1 or included therein At least 10, or at least 20, or at least 30, or at least 40, at least 50, or at least 60, or at least 70, of the DNA-tag sequences differ by 1 base in the PCR-Free tag sequence, or At least 80, or 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or all 161.
  • these PCR-Free tag sequences preferably include at least PCR-Free Index-1 to PCR-Free Index-10 in the 161 PCR-Free tag sequences shown in Table 1, or PCR-Free Index - 11 ⁇ PCR-Free Index-20, or PCR-Free Index-21 ⁇ PCR-Free Index-30, or PCR-Free Index-31 ⁇ PCR-Free Index-40, or PCR-Free Index-41 ⁇ PCR- Free Index-50, or PCR-Free Index-51 ⁇ PCR-Free Index-60, or PCR-Free Index-61 ⁇ PCR-Free Index-70, or PCR-Free Index-71 ⁇ PCR-Free Index-80, Or PCR-Free Index- 81 ⁇ PCR-Free Index-90, or PCR-Free Index-91 - PCR-Free Index-100, or PCR-Free Index-101 - PCR-Free Index-110, or PCR-Free Index -I ll ⁇ PCR-Free Index- 120, or PCR-Free Index
  • a difference of 1 base includes substitution, addition or deletion of 1 base in the tag sequence.
  • a DNA PCR-Free tag linker is also provided for use in DNA tag library construction and sequencing.
  • a DNA tag library constructed using the above DNA PCR-Free tag linker is also provided.
  • the present invention also provides a method of constructing a DNA tag library using the above oligonucleotide (DNA PCR-Free tag linker). Specifically, according to an embodiment of the present invention, referring to FIG. 2, the method includes:
  • the DNA template has two oligonucleotide strands.
  • the source of the DNA sample is not particularly limited and may be derived from all eukaryotic and prokaryotic Creature.
  • the DNA sample is a human DNA sample, and more specifically, may be a human genomic DNA sample.
  • the length of the DNA template is about 250 bp, thereby enabling further improvement in the efficiency of constructing the DNA tag library and subsequent sequencing. The inventors have found that with the method according to an embodiment of the present invention, a DNA tag library of a plurality of common model organisms can be efficiently constructed.
  • base A is added to the 3's ends of the two oligonucleotide strands of the DNA template.
  • a DNA template having a sticky end A was obtained.
  • the DNA template is subjected to end repair before the base A is added.
  • a linker containing one selected from the above-described group of isolated DNA tags according to an embodiment of the present invention is respectively connected to both ends of the DNA template having the sticky end A to obtain a ligation product.
  • the linker is one selected from the group of isolated oligonucleotides according to embodiments of the invention.
  • a DNA template having a sticky end A and a DNA PCR-Free tag linker are linked to DNA at the 3' end of both oligonucleotide strands of a DNA template having a sticky end A. Label joints are implemented.
  • the "ligation product" obtained according to the above embodiment of the present invention contains a target fragment, a DNA linker, and a DNA tag.
  • fragment of interest as used herein, the sequence of which corresponds to the sequence of the DNA template.
  • sequence of the target fragment corresponds to the sequence of the DNA template, which means that the sequence of the DNA template can be directly derived from the sequence of the target fragment, for example, the sequence of the target fragment can be identical to the sequence of the DNA template, It may be completely complementary, or even increase or decrease a known number of known bases, as long as the sequence of DNA can be obtained by limited calculation.
  • the ligation products obtained are isolated and recovered, and these ligation products constitute a DNA tag library.
  • the method of separating and recovering the linked product is not particularly limited, and those skilled in the art can select an appropriate method and apparatus for separation according to the characteristics of the linked product.
  • the ligation product can be separated and recovered by 2% agarose gel electrophoresis.
  • the present invention provides a method of constructing a DNA tag library, comprising:
  • DNA template preparation providing n DNA samples, n is an integer and an integer of 1 ⁇ n ⁇ 161, preferably n is an integer and 2 ⁇ n ⁇ 161 , the DNA sample can be from all eukaryotic and prokaryotic organisms, including Not limited to human DNA samples; preferably, according to an embodiment of the invention, the DNA template is 250 bp in length;

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Abstract

L'invention concerne des marqueurs d'ADN pour la construction d'une bibliothèque de marqueurs d'ADN, des oligonucléotides, des bibliothèques de marqueurs d'ADN et leur procédé de préparation, un procédé de détermination des informations de séquence d'un échantillon d'ADN, un procédé de détermination des informations de séquence d'une pluralité d'échantillons d'ADN, et un kit pour la construction des bibliothèques de marqueurs d'ADN. Lesdits marqueurs d'ADN sont constitués des nucléotides représentés dans la SEQ ID NO: (2N-1), N étant un entier quelconque de 1 à 161.
PCT/CN2011/079899 2010-09-21 2011-09-20 Marqueurs d'adn et leurs utilisations Ceased WO2012037877A1 (fr)

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CN201010299261XA CN101967476B (zh) 2010-09-21 2010-09-21 一种基于接头连接的DNA PCR-Free标签文库构建方法
CN201010299261.X 2010-09-21

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CN104562213A (zh) * 2014-12-26 2015-04-29 北京诺禾致源生物信息科技有限公司 扩增子文库及其构建方法
CN105401222A (zh) * 2015-12-30 2016-03-16 安诺优达基因科技(北京)有限公司 一种构建测序用dna文库的方法
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CN109689872B (zh) * 2016-11-21 2022-12-23 深圳华大智造科技股份有限公司 一种dna末端修复与加a的方法
CN108949905B (zh) * 2017-05-23 2022-05-17 深圳华大基因股份有限公司 对照文库及其构建方法
CN111748613A (zh) * 2019-03-27 2020-10-09 华大数极生物科技(深圳)有限公司 一种双标签接头设计方法及制备方法
CN112575388A (zh) * 2020-12-22 2021-03-30 深圳市睿法生物科技有限公司 一种单分子靶标基因建库方法及其试剂盒
CN112708619B (zh) * 2020-12-30 2022-05-17 纳昂达(南京)生物科技有限公司 Mgi平台的建库用接头、试剂盒及建库方法
CN112921107A (zh) * 2020-12-31 2021-06-08 深圳市慢性病防治中心(深圳市皮肤病防治研究所、深圳市肺部疾病防治研究所) 结核分枝杆菌异质性耐药检测方法、试剂盒及应用
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