WO2012037877A1 - Dna tags and use thereof - Google Patents

Dna tags and use thereof 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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Prior art keywords
dna
pcr
index
free
tag
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PCT/CN2011/079899
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French (fr)
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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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 WO2012037877A1 publication Critical patent/WO2012037877A1/en

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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

Provided are DNA tags for constructing a DNA tag library, oligonucleotides, DNA tag libraries and a method for preparing the same, a method for determining the sequence information of a DNA sample, a method for determining the sequence information of a plurality of DNA samples, and a kit for constructing the DNA tag libraries. Said DNA tags consist of the nucleotides shown in SEQ ID NO: (2N-1), in which N is any integer from 1 to 161.

Description

DNA标签及其应用 优先权信息  DNA tags and their applications
本申请请求 2010 年 9 月 21 日向中国国家知识产权局提交的、 专利申请号为 201010299261.X的专利申请的优先权和权益， 并且通过参照将其全文并入此处。  The present application claims priority to and the benefit of the patent application Serial No. 201010299261.X filed on Sep. 21, 2010, the disclosure of which is hereby incorporated by reference.
技术领域 Technical field
本发明涉及核酸测序技术领域， 特别是 DNA测序技术领域。 具体的， 本发明涉及 用于 DNA测序的 DNA标签及其应用。 更具体的， 本发明提供了用于构建 DNA标签文 库的 DNA标签、 寡核苷酸、 DNA标签文库及其制备方法、 确定 DNA样品序列信息的 方法、 确定多种 DNA样品序列信息的方法以及用于构建 DNA标签文库的试剂盒。 背景技术  The invention relates to the field of nucleic acid sequencing technology, in particular to the field of DNA sequencing technology. In particular, 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测序技术， 是重要的分子生物学分析方法之一， 它不仅为基因表达、 基因调 控等生物学基础研究提供重要数据， 而且也在疾病诊断学、基因治疗等应用研究中起着 重要的作用。基于 Solexa DNA测序平台（ Illumina ) , 釆用边合成边测序（ Sequencing By Synthesis, SBS ) , 具有所需样品量少， 高通量， 高精确性， 拥有筒单易操作的自动化 平台和功能强大等特点（例如参见 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 2-5kb samples for mate pair library sequencing; Illumina part#1005363 Rev.B , 通过参照将其全文并入本文） 。  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. . Based on the Solexa DNA Sequencing Platform (Illumina), Sequencing By Synthesis (SBS), with the required sample volume, high throughput, high accuracy, easy-to-operate automation platform and powerful functions, etc. Features (see, for example, 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.
然而， 目前对样品 DNA进行测序的方法， 仍有待改进。  However, the current method of sequencing sample DNA remains to be improved.
发明内容 Summary of the invention
本发明是基于发明人的下列发现而完成的：  The present invention has been completed based on the following findings of the inventors:
目前 Illumina公司基于 Solexa DNA测序平台推出了 DNA标签（也称为 index )建 库方法。 如图 1所示， 在 DNA标签建库流程中， 使用了 3条 PCR引物， 通过 PCR导 入标签来构建 DNA标签文库 ( Preparing samples for multiplexed Paired-End sequencing; Illumina part#1005361 Rev.B , 通过参照将其全文并入本文） 。 本申请的发明人发现， 上述标签文库制备方法存在着一些缺陷： 第一、 目前 Illumina公司只提供了 12种长度 为 6bp的标签序列， 标签的数量较少， 随着 Solexa测序通量的增加， 不能对大量样本 进行混合测序， 从而将浪费测序资源和影响到测序通量； 第二、 上述标签建库方法是通 过 PCR反应将标签序列导入到目的片段文库中的， 其对目的片段的 PCR扩增过程需要 釆用 3条 PCR引物 （两条公用 PCR引物和一条 PCR标签引物， 如图 1所示） ， 耗时 耗材， 且 PCR扩增效率不高。 第三、 上述标签建库方法中所釆用的接头不包含标签序 列， 因此对多种样品 DNA进行建库测序时， 各样品的标签文库需要独立构建， 即各自 分别通过 PCR反应来导入标签序列， 接着针对每一个标签文库分别切胶回收， 然后将 切胶回收所得的各个标签文库进行混合，最后才能将多种样品标签文库的混合物进行测 序， 不仅费时费力， 而且费用较高。  At present, 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. Third, 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.
本发明旨在解决现有技术问题的至少之一。 为此， 本发明的一个方面， 提出了一种 能够用于构建 DNA标签文库的 DNA标签（在本文中， 有时也筒单地称为 "标签" ） 。 根据本发明的一个方面， 本发明提出了一组分离的 DNA标签。 根据本发明的一些实施 例， 这些分离的 DNA标签由 SEQ ID NO: ( 2N-1 ) 所示的核苷酸构成， 其中 N=l-161 的任意整数。在本说明书中，这些 DNA标签分别被命名为 DNA Index-N,其中 N=l-161 的任意整数， 其序列如下表 1所示。 利用上述根据本发明实施例的 DNA标签， 通过将 DNA标签与样品 DNA或其等同物相连， 可以精确地表征 DNA的样品来源。 由此， 利 用上述 DNA标签， 可以同时构建多种样品的 DNA标签文库 （在本文中， 有时也称为 "标签文库" ；）， 从而可以通过将来源于不同样品的 DNA标签文库混合之后进行测序， 并且能够基于 DNA标签对 DNA标签文库的 DNA序列进行分类，从而可以获得多种样 品的 DNA序列信息，由此可以充分利用高通量的测序技术，例如利用 Solexa测序技术， 同时对多种 DNA标签文库进行测序， 从而提高 DNA标签文库的测序效率和通量。 发 明人惊奇地发现， 利用根据本发明实施例的 DNA标签构建 DNA标签文库， 能够精确 地对多种 DNA标签文库进行区分， 并且所得到的测序数据结果的稳定性和可重复性非 常好。 The present invention is directed to solving at least one of the problems of the prior art. To this end, in one aspect of the invention, a DNA tag (herein sometimes referred to as a "tag") that can be used to construct a library of DNA tags is proposed. According to one aspect of the invention, the invention proposes a set of isolated DNA tags. According to some embodiments of the invention, the isolated DNA tags are comprised of the nucleotides set forth in SEQ ID NO: (2N-1), wherein N = any integer from 1 to 161. In the present specification, these DNA tags are respectively named DNA Index-N, wherein N = any integer of 1-161, the sequence of which is shown in Table 1 below. Using the above-described DNA tag according to an embodiment of the present invention, the sample source of the DNA can be accurately characterized by linking the DNA tag to the sample DNA or its equivalent. Thus, by using the above DNA tag, 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. , And it is possible to classify DNA sequences of DNA tag libraries based on DNA tags, thereby obtaining DNA sequence information of various samples, thereby making full use of high-throughput sequencing technologies, such as using Solexa sequencing technology, and simultaneously screening multiple DNA tags. 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.
根据本发明的另一方面， 本发明还提供了用于将上述 DNA标签引入样品 DNA或其 等同物中的一组分离的寡核苷酸。根据本发明的实施例的一组分离的寡核苷酸， 具有第 一链和第二链， 所述第一链由 SEQ ID NO: 323所示的核苷酸构成， 所述第二链分别由 SEQ ID NO: ( 2N ) 所示的核苷酸构成， 其中 N=l-161的任意整数。 根据本发明的实施 例，这些寡核苷酸（在本说明书中，有时也称为 "DNA PCR-Free标签接头"、 "PCR-Free 标签接头" ）分别具有如前所述的根据本发明实施例的 DNA标签， 并且具有粘性末端 T, 因而， 可以通过连接反应， 将相应的 DNA标签引入到 DNA或其等同物中。 与 DNA标签 的命名方法类似， 在本说明书中， 与 DNA标签 DNA Index-N相对应的寡核苷酸 （ DNA PCR-Free标签接头）被命名为 DNA PCR-Free Index-N adapter,其中 N=l-161的任意整数， 进一步， DNA PCR-Free标签接头的第一链和第二链分别被命名为 DNA PCR-Free 接头 1.0和 PCR-Free Index-N, 其中 N=l-161的任意整数， 其序列如下表 1所示（表中所示序列 方向均是 5' - 3'方向） 。 根据本发明的实施例， 可以通过将 DNA PCR-Free 接头 1.0与 PCR-Free Index-N进行等摩尔退火处理而形成相应的具 Y型结构的 DNA PCR-Free标签 接头。  According to another aspect of the invention, the invention also provides a set of isolated oligonucleotides for introducing the above DNA tag into a sample DNA or an equivalent thereof. A set of isolated oligonucleotides according to an embodiment of the invention having a first strand and a second strand, said first strand being comprised of the nucleotides set forth in SEQ ID NO: 323, said second strand It is composed of the nucleotide represented by SEQ ID NO: (2N), wherein N = any integer of 1-161. According to an embodiment of the invention, 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. Similar to the method of naming DNA tags, in this specification, an oligonucleotide (DNA PCR-Free tag linker) corresponding to the DNA tag DNA Index-N is named DNA PCR-Free Index-N adapter, where N= Any integer of l-161, further, the first strand and the second strand of the DNA PCR-Free tag linker are named DNA PCR-Free linker 1.0 and PCR-Free Index-N, respectively, where N=l-161 of any integer The sequence is shown in Table 1 below (the sequence directions shown in the table are all 5' - 3' directions). According to an embodiment of the present invention, 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标签序列 （ DNA Index-N ) 及其相应的 DNA PCR-Free标签接头序列  DNA tag sequence (DNA Index-N) and its corresponding DNA PCR-Free tag linker sequence
Figure imgf000003_0001
DNA Index-8 CCTAACGT(15)
Figure imgf000003_0001
DNA Index-8 CCTAACGT(15)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTAACGTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTAACGTATCTCGTATGCCGTCTTCT
PCR-Free Index-8 PCR-Free Index-8
GCTTG(16)  GCTTG(16)
DNA Index-9 CACGTAGT(17)  DNA Index-9 CACGTAGT(17)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACGTAGTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACGTAGTATCTCGTATGCCGTCTTCT
PCR-Free Index-9 PCR-Free Index-9
GCTTG(18)  GCTTG (18)
DNA Index-10 GTAAGAGT(19)  DNA Index-10 GTAAGAGT(19)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTAAGAGTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTAAGAGTATCTCGTATGCCGTCTTCT
PCR-Free Index-10 PCR-Free Index-10
GCTTG(20)  GCTTG (20)
DNA Index-11 TACCTTCT(21)  DNA Index-11 TACCTTCT(21)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACCTTCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACCTTCTATCTCGTATGCCGTCTTCT
PCR-Free Index-11 PCR-Free Index-11
GCTTG(22)  GCTTG (22)
DNA Index-12 AAGTCTCT(23)  DNA Index-12 AAGTCTCT(23)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGTCTCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGTCTCTATCTCGTATGCCGTCTTCT
PCR-Free Index-12 PCR-Free Index-12
GCTTG(24)  GCTTG(24)
DNA Index-13 AGAGATCT(25)  DNA Index-13 AGAGATCT(25)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGAGATCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGAGATCTATCTCGTATGCCGTCTTCT
PCR-Free Index-13 PCR-Free Index-13
GCTTG(26)  GCTTG (26)
DNA Index-14 CCAGCGCT(27)  DNA Index-14 CCAGCGCT(27)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCAGCGCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCAGCGCTATCTCGTATGCCGTCTTCT
PCR-Free Index-14 PCR-Free Index-14
GCTTG(28)  GCTTG (28)
DNA Index-15 ATGAACCT(29)  DNA Index-15 ATGAACCT(29)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGAACCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGAACCTATCTCGTATGCCGTCTTCT
PCR-Free Index-15 PCR-Free Index-15
GCTTG(30)  GCTTG (30)
DNA Index-16 ACCAGACT(31)  DNA Index-16 ACCAGACT(31)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCAGACTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCAGACTATCTCGTATGCCGTCTTCT
PCR-Free Index-16 PCR-Free Index-16
GCTTG(32)  GCTTG (32)
DNA Index-17 CTATAACT(33)  DNA Index-17 CTATAACT(33)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTATAACTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTATAACTATCTCGTATGCCGTCTTCT
PCR-Free Index-17 PCR-Free Index-17
GCTTG(34)  GCTTG (34)
DNA Index-18 GCGGAACT(35)  DNA Index-18 GCGGAACT(35)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGGAACTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGGAACTATCTCGTATGCCGTCTTCT
PCR-Free Index-18 PCR-Free Index-18
GCTTG(36)  GCTTG (36)
DNA Index-19 CTAGTTAT(37)  DNA Index-19 CTAGTTAT(37)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAGTTATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAGTTATATCTCGTATGCCGTCTTCT
PCR-Free Index-19 PCR-Free Index-19
GCTTG(38)  GCTTG (38)
DNA Index-20 TCTTATAT(39)  DNA Index-20 TCTTATAT(39)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCTTATATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCTTATATATCTCGTATGCCGTCTTCT
PCR-Free Index-20 PCR-Free Index-20
GCTTG(40)  GCTTG (40)
DNA Index-21 GAATCGAT(41)  DNA Index-21 GAATCGAT(41)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAATCGATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAATCGATATCTCGTATGCCGTCTTCT
PCR-Free Index-21 PCR-Free Index-21
GCTTG(42)  GCTTG (42)
DNA Index-22 AATAAGAT(43)  DNA Index-22 AATAAGAT(43)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAATAAGATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAATAAGATATCTCGTATGCCGTCTTCT
PCR-Free Index-22 PCR-Free Index-22
GCTTG(44) DNA Index-23 TATGCCAT(45) GCTTG (44) DNA Index-23 TATGCCAT(45)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATGCCATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATGCCATATCTCGTATGCCGTCTTCT
PCR-Free Index-23 PCR-Free Index-23
GCTTG(46)  GCTTG (46)
DNA Index-24 ATTCTAAT(47)  DNA Index-24 ATTCTAAT(47)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCTAATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCTAATATCTCGTATGCCGTCTTCT
PCR-Free Index-24 PCR-Free Index-24
GCTTG(48)  GCTTG (48)
DNA Index-25 TAATGTTG(49)  DNA Index-25 TAATGTTG(49)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATGTTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATGTTGATCTCGTATGCCGTCTTCT
PCR-Free Index-25 PCR-Free Index-25
GCTTG(50)  GCTTG (50)
DNA Index-26 GTTACTTG(51)  DNA Index-26 GTTACTTG(51)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTACTTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTACTTGATCTCGTATGCCGTCTTCT
PCR-Free Index-26 PCR-Free Index-26
GCTTG(52)  GCTTG (52)
DNA Index-27 ATTCACTG(53)  DNA Index-27 ATTCACTG(53)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCACTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCACTGATCTCGTATGCCGTCTTCT
PCR-Free Index-27 PCR-Free Index-27
GCTTG(54)  GCTTG (54)
DNA Index-28 ATCATATG(55)  DNA Index-28 ATCATATG(55)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCATATGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCATATGATCTCGTATGCCGTCTTCT
PCR-Free Index-28 PCR-Free Index-28
GCTTG(56)  GCTTG(56)
DNA Index-29 GCTTAATG(57)  DNA Index-29 GCTTAATG(57)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCTTAATGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCTTAATGATCTCGTATGCCGTCTTCT
PCR-Free Index-29 PCR-Free Index-29
GCTTG(58)  GCTTG (58)
DNA Index-30 GGATATGG(59)  DNA Index-30 GGATATGG(59)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGATATGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGATATGGATCTCGTATGCCGTCTTCT
PCR-Free Index-30 PCR-Free Index-30
GCTTG(60)  GCTTG (60)
DNA Index-31 CTTGATGG(61)  DNA Index-31 CTTGATGG(61)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGATGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGATGGATCTCGTATGCCGTCTTCT
PCR-Free Index-31 PCR-Free Index-31
GCTTG(62)  GCTTG (62)
DNA Index-32 AAGATCGG(63)  DNA Index-32 AAGATCGG(63)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGATCGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGATCGGATCTCGTATGCCGTCTTCT
PCR-Free Index-32 PCR-Free Index-32
GCTTG(64)  GCTTG (64)
DNA Index-33 TTAACCGG(65)  DNA Index-33 TTAACCGG(65)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAACCGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAACCGGATCTCGTATGCCGTCTTCT
PCR-Free Index-33 PCR-Free Index-33
GCTTG(66)  GCTTG (66)
DNA Index-34 CTAAGTCG(67)  DNA Index-34 CTAAGTCG(67)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAAGTCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAAGTCGATCTCGTATGCCGTCTTCT
PCR-Free Index-34 PCR-Free Index-34
GCTTG(68)  GCTTG (68)
DNA Index-35 TATTCGCG(69)  DNA Index-35 TATTCGCG(69)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATTCGCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATTCGCGATCTCGTATGCCGTCTTCT
PCR-Free Index-35 PCR-Free Index-35
GCTTG(70)  GCTTG (70)
DNA Index-36 GAAGCACG(71)  DNA Index-36 GAAGCACG(71)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAAGCACGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAAGCACGATCTCGTATGCCGTCTTCT
PCR-Free Index-36 PCR-Free Index-36
GCTTG(72)  GCTTG (72)
DNA Index-37 TCCAGTAG(73)  DNA Index-37 TCCAGTAG(73)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCAGTAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCAGTAGATCTCGTATGCCGTCTTCT
PCR-Free Index-37 PCR-Free Index-37
GCTTG(74) DNA Index-38 TTGTCTAG(75) GCTTG (74) DNA Index-38 TTGTCTAG(75)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGTCTAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGTCTAGATCTCGTATGCCGTCTTCT
PCR-Free Index-38 PCR-Free Index-38
GCTTG(76)  GCTTG (76)
DNA Index-39 AGCGCTAG(77)  DNA Index-39 AGCGCTAG(77)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCGCTAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCGCTAGATCTCGTATGCCGTCTTCT
PCR-Free Index-39 PCR-Free Index-39
GCTTG(78)  GCTTG (78)
DNA Index-40 CCTGTGAG(79)  DNA Index-40 CCTGTGAG(79)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTGTGAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTGTGAGATCTCGTATGCCGTCTTCT
PCR-Free Index-40 PCR-Free Index-40
GCTTG(80)  GCTTG (80)
DNA Index-41 CAACTAAG(81)  DNA Index-41 CAACTAAG(81)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAACTAAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAACTAAGATCTCGTATGCCGTCTTCT
PCR-Free Index-41 PCR-Free Index-41
GCTTG(82)  GCTTG (82)
DNA Index-42 ATAGGAAG(83)  DNA Index-42 ATAGGAAG(83)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATAGGAAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATAGGAAGATCTCGTATGCCGTCTTCT
PCR-Free Index-42 PCR-Free Index-42
GCTTG(84)  GCTTG (84)
DNA Index-43 ACTACAAG(85)  DNA Index-43 ACTACAAG(85)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTACAAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTACAAGATCTCGTATGCCGTCTTCT
PCR-Free Index-43 PCR-Free Index-43
GCTTG(86)  GCTTG (86)
DNA Index-44 GATGGTTC(87)  DNA Index-44 GATGGTTC(87)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATGGTTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATGGTTCATCTCGTATGCCGTCTTCT
PCR-Free Index-44 PCR-Free Index-44
GCTTG(88)  GCTTG (88)
DNA Index-45 CCACATTC(89)  DNA Index-45 CCACATTC(89)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCACATTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCACATTCATCTCGTATGCCGTCTTCT
PCR-Free Index-45 PCR-Free Index-45
GCTTG(90)  GCTTG (90)
DNA Index-46 TCTTGGTC(91)  DNA Index-46 TCTTGGTC(91)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCTTGGTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCTTGGTCATCTCGTATGCCGTCTTCT
PCR-Free Index-46 PCR-Free Index-46
GCTTG(92)  GCTTG (92)
DNA Index-47 CGAGGATC(93)  DNA Index-47 CGAGGATC(93)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGAGGATCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGAGGATCATCTCGTATGCCGTCTTCT
PCR-Free Index-47 PCR-Free Index-47
GCTTG(94)  GCTTG (94)
DNA Index-48 AGTCCATC(95)  DNA Index-48 AGTCCATC(95)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTCCATCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTCCATCATCTCGTATGCCGTCTTCT
PCR-Free Index-48 PCR-Free Index-48
GCTTG(96)  GCTTG (96)
DNA Index-49 CACTAATC(97)  DNA Index-49 CACTAATC(97)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAATCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAATCATCTCGTATGCCGTCTTCT
PCR-Free Index-49 PCR-Free Index-49
GCTTG(98)  GCTTG (98)
DNA Index-50 TAAGGCGC(99)  DNA Index-50 TAAGGCGC(99)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAAGGCGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAAGGCGCATCTCGTATGCCGTCTTCT
PCR-Free Index-50 PCR-Free Index-50
GCTTG(IOO)  GCTTG (IOO)
DNA Index-51 AATAGAGC(lOl)  DNA Index-51 AATAGAGC(lOl)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAATAGAGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAATAGAGCATCTCGTATGCCGTCTTCT
PCR-Free Index-51 PCR-Free Index-51
GCTTG(102)  GCTTG (102)
DNA Index-52 ACTGTTCC(103)  DNA Index-52 ACTGTTCC(103)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTGTTCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTGTTCCATCTCGTATGCCGTCTTCT
PCR-Free Index-52 PCR-Free Index-52
GCTTG(104) DNA Index-53 CTTCCTCC(IOS) GCTTG (104) DNA Index-53 CTTCCTCC (IOS)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTCCTCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTCCTCCATCTCGTATGCCGTCTTCT
PCR-Free Index-53 PCR-Free Index-53
GCTTG(106)  GCTTG (106)
DNA Index-54 GCGACTCC(107)  DNA Index-54 GCGACTCC(107)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGACTCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGACTCCATCTCGTATGCCGTCTTCT
PCR-Free Index-54 PCR-Free Index-54
GCTTG(108)  GCTTG (108)
DNA Index-55 TACAGGCC(109)  DNA Index-55 TACAGGCC(109)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACAGGCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACAGGCCATCTCGTATGCCGTCTTCT
PCR-Free Index-55 PCR-Free Index-55
GCTTG(llO)  GCTTG (llO)
DNA Index-56 GTTAAGCC(lll)  DNA Index-56 GTTAAGCC(lll)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTAAGCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTAAGCCATCTCGTATGCCGTCTTCT
PCR-Free Index-56 PCR-Free Index-56
GCTTG(112)  GCTTG (112)
DNA Index-57 TAATTACC(113)  DNA Index-57 TAATTACC(113)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATTACCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATTACCATCTCGTATGCCGTCTTCT
PCR-Free Index-57 PCR-Free Index-57
GCTTG(114)  GCTTG (114)
DNA Index-58 ATAACACC(115)  DNA Index-58 ATAACACC(115)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATAACACCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATAACACCATCTCGTATGCCGTCTTCT
PCR-Free Index-58 PCR-Free Index-58
GCTTG(116)  GCTTG (116)
DNA Index-59 CGTAGGAC(117)  DNA Index-59 CGTAGGAC(117)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTAGGACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTAGGACATCTCGTATGCCGTCTTCT
PCR-Free Index-59 PCR-Free Index-59
GCTTG(118)  GCTTG (118)
DNA Index-60 CTCTCGAC(119)  DNA Index-60 CTCTCGAC(119)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCTCGACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCTCGACATCTCGTATGCCGTCTTCT
PCR-Free Index-60 PCR-Free Index-60
GCTTG(120)  GCTTG (120)
DNA Index-61 CTACGCAC(121)  DNA Index-61 CTACGCAC(121)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTACGCACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTACGCACATCTCGTATGCCGTCTTCT
PCR-Free Index-61 PCR-Free Index-61
GCTTG(122)  GCTTG (122)
DNA Index-62 AGGTTAAC(123)  DNA Index-62 AGGTTAAC(123)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGGTTAACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGGTTAACATCTCGTATGCCGTCTTCT
PCR-Free Index-62 PCR-Free Index-62
GCTTG(124)  GCTTG (124)
DNA Index-63 GTTGCAAC(125)  DNA Index-63 GTTGCAAC(125)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTGCAACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTGCAACATCTCGTATGCCGTCTTCT
PCR-Free Index-63 PCR-Free Index-63
GCTTG(126)  GCTTG (126)
DNA Index-64 CTCAATTA(127)  DNA Index-64 CTCAATTA(127)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCAATTAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCAATTAATCTCGTATGCCGTCTTCT
PCR-Free Index-64 PCR-Free Index-64
GCTTG(128)  GCTTG (128)
DNA Index-65 CAAGTCTA(129)  DNA Index-65 CAAGTCTA(129)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAAGTCTAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAAGTCTAATCTCGTATGCCGTCTTCT
PCR-Free Index-65 PCR-Free Index-65
GCTTG(130)  GCTTG (130)
DNA Index-66 ACAACCTA(131)  DNA Index-66 ACAACCTA(131)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAACCTAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAACCTAATCTCGTATGCCGTCTTCT
PCR-Free Index-66 PCR-Free Index-66
GCTTG(132)  GCTTG (132)
DNA Index-67 CTACCATA(133)  DNA Index-67 CTACCATA(133)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTACCATAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTACCATAATCTCGTATGCCGTCTTCT
PCR-Free Index-67 PCR-Free Index-67
GCTTG(134) DNA Index-68 GACACATA(135) GCTTG (134) DNA Index-68 GACACATA(135)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACACATAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACACATAATCTCGTATGCCGTCTTCT
PCR-Free Index-68 PCR-Free Index-68
GCTTG(136)  GCTTG (136)
DNA Index-69 AGATAATA(137)  DNA Index-69 AGATAATA(137)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGATAATAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGATAATAATCTCGTATGCCGTCTTCT
PCR-Free Index-69 PCR-Free Index-69
GCTTG(138)  GCTTG (138)
DNA Index-70 CGCGGTGA(139)  DNA Index-70 CGCGGTGA(139)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGCGGTGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGCGGTGAATCTCGTATGCCGTCTTCT
PCR-Free Index-70 PCR-Free Index-70
GCTTG(140)  GCTTG (140)
DNA Index-71 TACTATGA(141)  DNA Index-71 TACTATGA(141)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACTATGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACTATGAATCTCGTATGCCGTCTTCT
PCR-Free Index-71 PCR-Free Index-71
GCTTG(142)  GCTTG (142)
DNA Index-72 TTGTTGGA(143)  DNA Index-72 TTGTTGGA(143)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGTTGGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGTTGGAATCTCGTATGCCGTCTTCT
PCR-Free Index-72 PCR-Free Index-72
GCTTG(144)  GCTTG (144)
DNA Index-73 AGTGAGGA(145)  DNA Index-73 AGTGAGGA(145)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTGAGGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTGAGGAATCTCGTATGCCGTCTTCT
PCR-Free Index-73 PCR-Free Index-73
GCTTG(146)  GCTTG (146)
DNA Index-74 ATCGCCGA(147)  DNA Index-74 ATCGCCGA(147)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCGCCGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCGCCGAATCTCGTATGCCGTCTTCT
PCR-Free Index-74 PCR-Free Index-74
GCTTG(148)  GCTTG (148)
DNA Index-75 CTTATAGA(149)  DNA Index-75 CTTATAGA(149)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTATAGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTATAGAATCTCGTATGCCGTCTTCT
PCR-Free Index-75 PCR-Free Index-75
GCTTG(150)  GCTTG (150)
DNA Index-76 CCATGAGA(lSl)  DNA Index-76 CCATGAGA(lSl)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCATGAGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCATGAGAATCTCGTATGCCGTCTTCT
PCR-Free Index-76 PCR-Free Index-76
GCTTG(152)  GCTTG (152)
DNA Index-77 TCACCTCA(153)  DNA Index-77 TCACCTCA(153)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCACCTCAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCACCTCAATCTCGTATGCCGTCTTCT
PCR-Free Index-77 PCR-Free Index-77
GCTTG(154)  GCTTG (154)
DNA Index-78 ACCTTGCA(155)  DNA Index-78 ACCTTGCA (155)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCTTGCAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCTTGCAATCTCGTATGCCGTCTTCT
PCR-Free Index-78 PCR-Free Index-78
GCTTG(156)  GCTTG (156)
DNA Index-79 ATACTCCA(157)  DNA Index-79 ATACTCCA(157)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATACTCCAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATACTCCAATCTCGTATGCCGTCTTCT
PCR-Free Index-79 PCR-Free Index-79
GCTTG(158)  GCTTG (158)
DNA Index-80 GTTCGACA(159)  DNA Index-80 GTTCGACA (159)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTCGACAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTCGACAATCTCGTATGCCGTCTTCT
PCR-Free Index-80 PCR-Free Index-80
GCTTG(160)  GCTTG (160)
DNA Index-81 CATCATAA(161)  DNA Index-81 CATCATAA(161)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATCATAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATCATAAATCTCGTATGCCGTCTTCT
PCR-Free Index-81 PCR-Free Index-81
GCTTG(162)  GCTTG (162)
DNA Index-82 CACATGAA(163)  DNA Index-82 CACATGAA(163)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACATGAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACATGAAATCTCGTATGCCGTCTTCT
PCR-Free Index-82 PCR-Free Index-82
GCTTG(164) DNA Index-83 ATGAGGAA(165) GCTTG (164) DNA Index-83 ATGAGGAA(165)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGAGGAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGAGGAAATCTCGTATGCCGTCTTCT
PCR-Free Index-83 PCR-Free Index-83
GCTTG(166)  GCTTG (166)
DNA Index-84 TCCTCCAA(167)  DNA Index-84 TCCTCCAA(167)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCTCCAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCTCCAAATCTCGTATGCCGTCTTCT
PCR-Free Index-84 PCR-Free Index-84
GCTTG(168)  GCTTG (168)
DNA Index-85 TTAGACAA(169)  DNA Index-85 TTAGACAA(169)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGACAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGACAAATCTCGTATGCCGTCTTCT
PCR-Free Index-85 PCR-Free Index-85
GCTTG(170)  GCTTG (170)
DNA Index-86 GTCCAGAA(171)  DNA Index-86 GTCCAGAA(171)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCCAGAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCCAGAAATCTCGTATGCCGTCTTCT
PCR-Free Index-86 PCR-Free Index-86
GCTTG(172)  GCTTG (172)
DNA Index-87 ATCTATCG(173)  DNA Index-87 ATCTATCG(173)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTATCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTATCGATCTCGTATGCCGTCTTCT
PCR-Free Index-87 PCR-Free Index-87
GCTTG(174)  GCTTG (174)
DNA Index-88 TTACTGTT(175)  DNA Index-88 TTACTGTT(175)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTACTGTTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTACTGTTATCTCGTATGCCGTCTTCT
PCR-Free Index-88 PCR-Free Index-88
GCTTG(176)  GCTTG (176)
DNA Index-89 ACACGCGG(177)  DNA Index-89 ACACGCGG(177)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACACGCGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACACGCGGATCTCGTATGCCGTCTTCT
PCR-Free Index-89 PCR-Free Index-89
GCTTG(178)  GCTTG (178)
DNA Index-90 TATCCAGA(179)  DNA Index-90 TATCCAGA(179)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATCCAGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATCCAGAATCTCGTATGCCGTCTTCT
PCR-Free Index-90 PCR-Free Index-90
GCTTG(180)  GCTTG (180)
DNA Index-91 TAGGAATA(181)  DNA Index-91 TAGGAATA(181)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGGAATAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGGAATAATCTCGTATGCCGTCTTCT
PCR-Free Index-91 PCR-Free Index-91
GCTTG(182)  GCTTG (182)
DNA Index-92 GAACGTGA(183)  DNA Index-92 GAACGTGA(183)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAACGTGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAACGTGAATCTCGTATGCCGTCTTCT
PCR-Free Index-92 PCR-Free Index-92
GCTTG(184)  GCTTG (184)
DNA Index-93 CCGCACAG(185)  DNA Index-93 CCGCACAG(185)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGCACAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGCACAGATCTCGTATGCCGTCTTCT
PCR-Free Index-93 PCR-Free Index-93
GCTTG(186)  GCTTG (186)
DNA Index-94 ATTGCGTT(187)  DNA Index-94 ATTGCGTT (187)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTGCGTTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTGCGTTATCTCGTATGCCGTCTTCT
PCR-Free Index-94 PCR-Free Index-94
GCTTG(188)  GCTTG (188)
DNA Index-95 TCGTAAGC(189)  DNA Index-95 TCGTAAGC(189)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGTAAGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGTAAGCATCTCGTATGCCGTCTTCT
PCR-Free Index-95 PCR-Free Index-95
GCTTG(190)  GCTTG (190)
DNA Index-96 CCGTCACG(191)  DNA Index-96 CCGTCACG(191)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCACGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCACGATCTCGTATGCCGTCTTCT
PCR-Free Index-96 PCR-Free Index-96
GCTTG(192)  GCTTG (192)
DNA Index-97 GCGAAGTA(193)  DNA Index-97 GCGAAGTA(193)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGAAGTAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGAAGTAATCTCGTATGCCGTCTTCT
PCR-Free Index-97 PCR-Free Index-97
GCTTG(194) DNA Index-98 GGACTGCG(195) GCTTG (194) DNA Index-98 GGACTGCG(195)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGACTGCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGACTGCGATCTCGTATGCCGTCTTCT
PCR-Free Index-98 PCR-Free Index-98
GCTTG(196)  GCTTG (196)
DNA Index-99 GAGCATTG(197)  DNA Index-99 GAGCATTG (197)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGCATTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGCATTGATCTCGTATGCCGTCTTCT
PCR-Free Index-99 PCR-Free Index-99
GCTTG(198)  GCTTG (198)
DNA Index-100 TCGCCGTG(199)  DNA Index-100 TCGCCGTG(199)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGCCGTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGCCGTGATCTCGTATGCCGTCTTCT
PCR-Free Index-100 PCR-Free Index-100
GCTTG(200)  GCTTG (200)
DNA Index-101 CAGCGGCG(201)  DNA Index-101 CAGCGGCG(201)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGCGGCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGCGGCGATCTCGTATGCCGTCTTCT
PCR-Free Index-qOl PCR-Free Index-qOl
GCTTG(202)  GCTTG (202)
DNA Index-102 AAGGATGC(203)  DNA Index-102 AAGGATGC(203)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGGATGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGGATGCATCTCGTATGCCGTCTTCT
PCR-Free Index-102 PCR-Free Index-102
GCTTG(204)  GCTTG (204)
DNA Index-103 GCAATGGC(205)  DNA Index-103 GCAATGGC(205)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCAATGGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCAATGGCATCTCGTATGCCGTCTTCT
PCR-Free Index-103 PCR-Free Index-103
GCTTG(206)  GCTTG (206)
DNA Index-104 GTATTCTC(207)  DNA Index-104 GTATTCTC (207)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTATTCTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTATTCTCATCTCGTATGCCGTCTTCT
PCR-Free Index-104 PCR-Free Index-104
GCTTG(208)  GCTTG (208)
DNA Index-105 GTCATTAC(209)  DNA Index-105 GTCATTAC(209)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCATTACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCATTACATCTCGTATGCCGTCTTCT
PCR-Free Index-105 PCR-Free Index-105
GCTTG(210)  GCTTG (210)
DNA Index-106 ATCCAAGC(211)  DNA Index-106 ATCCAAGC(211)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCCAAGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCCAAGCATCTCGTATGCCGTCTTCT
PCR-Free Index-106 PCR-Free Index-106
GCTTG(212)  GCTTG (212)
DNA Index-107 GGTATACT(213)  DNA Index-107 GGTATACT(213)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGTATACTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGTATACTATCTCGTATGCCGTCTTCT
PCR-Free Index-107 PCR-Free Index-107
GCTTG(214)  GCTTG (214)
DNA Index-108 TTGCGTGC(215)  DNA Index-108 TTGCGTGC(215)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGCGTGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGCGTGCATCTCGTATGCCGTCTTCT
PCR-Free Index-108 PCR-Free Index-108
GCTTG(216)  GCTTG (216)
DNA Index-109 TCCGACGG(217)  DNA Index-109 TCCGACGG(217)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCGACGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCGACGGATCTCGTATGCCGTCTTCT
PCR-Free Index-109 PCR-Free Index-109
GCTTG(218)  GCTTG (218)
DNA Index-110 GCAGGCAT(219)  DNA Index-110 GCAGGCAT(219)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCAGGCATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCAGGCATATCTCGTATGCCGTCTTCT
PCR-Free Index-110 PCR-Free Index-110
GCTTG(220)  GCTTG (220)
DNA Index-Ill GCCAGCGA(221)  DNA Index-Ill GCCAGCGA(221)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAGCGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAGCGAATCTCGTATGCCGTCTTCT
PCR-Free Index-Ill PCR-Free Index-Ill
GCTTG(222)  GCTTG (222)
DNA Index-112 CACACTGG(223)  DNA Index-112 CACACTGG(223)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACACTGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACACTGGATCTCGTATGCCGTCTTCT
PCR-Free Index-112 PCR-Free Index-112
GCTTG(224) DNA Index-113 GGCCTCGC(225) GCTTG (224) DNA Index-113 GGCCTCGC(225)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCCTCGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCCTCGCATCTCGTATGCCGTCTTCT
PCR-Free Index-113 PCR-Free Index-113
GCTTG(226)  GCTTG (226)
DNA Index-114 GGCGCGCA(227)  DNA Index-114 GGCGCGCA (227)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCGCGCAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCGCGCAATCTCGTATGCCGTCTTCT
PCR-Free Index-114 PCR-Free Index-114
GCTTG(228)  GCTTG (228)
DNA Index-115 CGCCACCT(229)  DNA Index-115 CGCCACCT(229)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGCCACCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGCCACCTATCTCGTATGCCGTCTTCT
PCR-Free Index-115 PCR-Free Index-115
GCTTG(230)  GCTTG (230)
DNA Index-116 CATGCGGC(231)  DNA Index-116 CATGCGGC(231)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATGCGGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATGCGGCATCTCGTATGCCGTCTTCT
PCR-Free Index-116 PCR-Free Index-116
GCTTG(232)  GCTTG (232)
DNA Index-117 GGCAACAG(233)  DNA Index-117 GGCAACAG(233)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCAACAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCAACAGATCTCGTATGCCGTCTTCT
PCR-Free Index-117 PCR-Free Index-117
GCTTG(234)  GCTTG (234)
DNA Index-118 CGGTATCA(235)  DNA Index-118 CGGTATCA(235)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGTATCAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGTATCAATCTCGTATGCCGTCTTCT
PCR-Free Index-118 PCR-Free Index-118
GCTTG(236)  GCTTG (236)
DNA Index-119 CGGCCAAT(237)  DNA Index-119 CGGCCAAT(237)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGCCAATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGCCAATATCTCGTATGCCGTCTTCT
PCR-Free Index-119 PCR-Free Index-119
GCTTG(238)  GCTTG (238)
DNA Index-120 AGCCGTCC(239)  DNA Index-120 AGCCGTCC(239)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCCGTCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCCGTCCATCTCGTATGCCGTCTTCT
PCR-Free Index-120 PCR-Free Index-120
GCTTG(240)  GCTTG (240)
DNA Index-121 ACAGAGTG(241)  DNA Index-121 ACAGAGTG(241)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGAGTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGAGTGATCTCGTATGCCGTCTTCT
PCR-Free Index-121 PCR-Free Index-121
GCTTG(242)  GCTTG (242)
DNA Index-122 ACGCAGCC(243)  DNA Index-122 ACGCAGCC(243)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACGCAGCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACGCAGCCATCTCGTATGCCGTCTTCT
PCR-Free Index-122 PCR-Free Index-122
GCTTG(244)  GCTTG (244)
DNA Index-123 GAGCTGAC(245)  DNA Index-123 GAGCTGAC(245)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGCTGACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGCTGACATCTCGTATGCCGTCTTCT
PCR-Free Index-123 PCR-Free Index-123
GCTTG(246)  GCTTG (246)
DNA Index-124 TGATGGCT(247)  DNA Index-124 TGATGGCT(247)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGATGGCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGATGGCTATCTCGTATGCCGTCTTCT
PCR-Free Index-124 PCR-Free Index-124
GCTTG(248)  GCTTG (248)
DNA Index-125 TGAATCAT(249)  DNA Index-125 TGAATCAT(249)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGAATCATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGAATCATATCTCGTATGCCGTCTTCT
PCR-Free Index-125 PCR-Free Index-125
GCTTG(250)  GCTTG (250)
DNA Index-126 TGACAGAC(251)  DNA Index-126 TGACAGAC(251)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACAGACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACAGACATCTCGTATGCCGTCTTCT
PCR-Free Index-126 PCR-Free Index-126
GCTTG(252)  GCTTG (252)
DNA Index-127 GTGGTCGT(253)  DNA Index-127 GTGGTCGT (253)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGTCGTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGTCGTATCTCGTATGCCGTCTTCT
PCR-Free Index-127 PCR-Free Index-127
GCTTG(254) DNA Index-128 GCGTGGAG(255) GCTTG (254) DNA Index-128 GCGTGGAG(255)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGTGGAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGTGGAGATCTCGTATGCCGTCTTCT
PCR-Free Index-128 PCR-Free Index-128
GCTTG(256)  GCTTG (256)
DNA Index-129 ACTTCCGC(257)  DNA Index-129 ACTTCCGC (257)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTCCGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTCCGCATCTCGTATGCCGTCTTCT
PCR-Free Index-129 PCR-Free Index-129
GCTTG(258)  GCTTG (258)
DNA Index-130 ACATGTAC(259)  DNA Index-130 ACATGTAC(259)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACATGTACATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACATGTACATCTCGTATGCCGTCTTCT
PCR-Free Index-130 PCR-Free Index-130
GCTTG(260)  GCTTG (260)
DNA Index-131 CCGGCTAA(261)  DNA Index-131 CCGGCTAA(261)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGGCTAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGGCTAAATCTCGTATGCCGTCTTCT
PCR-Free Index-131 PCR-Free Index-131
GCTTG(262)  GCTTG (262)
DNA Index-132 CGATCCTG(263)  DNA Index-132 CGATCCTG (263)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATCCTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATCCTGATCTCGTATGCCGTCTTCT
PCR-Free Index-132 PCR-Free Index-132
GCTTG(264)  GCTTG (264)
DNA Index-133 GACGATAT(265)  DNA Index-133 GACGATAT(265)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACGATATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACGATATATCTCGTATGCCGTCTTCT
PCR-Free Index-133 PCR-Free Index-133
GCTTG(266)  GCTTG (266)
DNA Index-134 CCTGGCCA(267)  DNA Index-134 CCTGGCCA (267)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTGGCCAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTGGCCAATCTCGTATGCCGTCTTCT
PCR-Free Index-134 PCR-Free Index-134
GCTTG(268)  GCTTG (268)
DNA Index-135 AAGACGTC(269)  DNA Index-135 AAGACGTC (269)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGACGTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGACGTCATCTCGTATGCCGTCTTCT
PCR-Free Index-135 PCR-Free Index-135
GCTTG(270)  GCTTG (270)
DNA Index-136 GCTCTCTA(271)  DNA Index-136 GCTCTCTA(271)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCTCTCTAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCTCTCTAATCTCGTATGCCGTCTTCT
PCR-Free Index-136 PCR-Free Index-136
GCTTG(272)  GCTTG (272)
DNA Index-137 AGCGTGTC(273)  DNA Index-137 AGCGTGTC (273)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCGTGTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCGTGTCATCTCGTATGCCGTCTTCT
PCR-Free Index-137 PCR-Free Index-137
GCTTG(274)  GCTTG (274)
DNA Index-138 CCGTTGTT(275)  DNA Index-138 CCGTTGTT(275)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTTGTTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTTGTTATCTCGTATGCCGTCTTCT
PCR-Free Index-138 PCR-Free Index-138
GCTTG(276)  GCTTG (276)
DNA Index-139 TTGCTACG(277)  DNA Index-139 TTGCTACG(277)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGCTACGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGCTACGATCTCGTATGCCGTCTTCT
PCR-Free Index-139 PCR-Free Index-139
GCTTG(278)  GCTTG (278)
DNA Index-140 TGTAACCA(279)  DNA Index-140 TGTAACCA (279)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGTAACCAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGTAACCAATCTCGTATGCCGTCTTCT
PCR-Free Index-140 PCR-Free Index-140
GCTTG(280)  GCTTG (280)
DNA Index-141 TGTGTTAA(281)  DNA Index-141 TGTGTTAA(281)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGTGTTAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGTGTTAAATCTCGTATGCCGTCTTCT
PCR-Free Index-141 PCR-Free Index-141
GCTTG(282)  GCTTG (282)
DNA Index-142 GATAGCCG(283)  DNA Index-142 GATAGCCG(283)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATAGCCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATAGCCGATCTCGTATGCCGTCTTCT
PCR-Free Index-142 PCR-Free Index-142
GCTTG(284) DNA Index-143 TAACACCG(285) GCTTG (284) DNA Index-143 TAACACCG(285)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAACACCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAACACCGATCTCGTATGCCGTCTTCT
PCR-Free Index-143 PCR-Free Index-143
GCTTG(286)  GCTTG (286)
DNA Index-144 AGTAGTTA(287)  DNA Index-144 AGTAGTTA(287)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTAGTTAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTAGTTAATCTCGTATGCCGTCTTCT
PCR-Free Index-144 PCR-Free Index-144
GCTTG(288)  GCTTG (288)
DNA Index-145 GTCTGCCT(289)  DNA Index-145 GTCTGCCT(289)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCTGCCTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCTGCCTATCTCGTATGCCGTCTTCT
PCR-Free Index-145 PCR-Free Index-145
GCTTG(290)  GCTTG (290)
DNA Index-146 GGAGTAGA(291)  DNA Index-146 GGAGTAGA(291)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGAGTAGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGAGTAGAATCTCGTATGCCGTCTTCT
PCR-Free Index-146 PCR-Free Index-146
GCTTG(292)  GCTTG (292)
DNA Index-147 TGCGCAGC(293)  DNA Index-147 TGCGCAGC(293)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCGCAGCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCGCAGCATCTCGTATGCCGTCTTCT
PCR-Free Index-147 PCR-Free Index-147
GCTTG(294)  GCTTG (294)
DNA Index-148 TGCCTATA(295)  DNA Index-148 TGCCTATA(295)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCCTATAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCCTATAATCTCGTATGCCGTCTTCT
PCR-Free Index-148 PCR-Free Index-148
GCTTG(296)  GCTTG (296)
DNA Index-149 TGCTAGTG(297)  DNA Index-149 TGCTAGTG(297)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCTAGTGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCTAGTGATCTCGTATGCCGTCTTCT
PCR-Free Index-149 PCR-Free Index-149
GCTTG(298)  GCTTG (298)
DNA Index-150 CCGAGCTC(299)  DNA Index-150 CCGAGCTC(299)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGAGCTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGAGCTCATCTCGTATGCCGTCTTCT
PCR-Free Index-150 PCR-Free Index-150
GCTTG(300)  GCTTG (300)
DNA Index-151 CGGATTAG(301)  DNA Index-151 CGGATTAG(301)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGATTAGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGATTAGATCTCGTATGCCGTCTTCT
PCR-Free Index-151 PCR-Free Index-151
GCTTG(302)  GCTTG (302)
DNA Index-152 CGGACGGA(303)  DNA Index-152 CGGACGGA(303)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGACGGAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGACGGAATCTCGTATGCCGTCTTCT
PCR-Free Index-152 PCR-Free Index-152
GCTTG(304)  GCTTG (304)
DNA Index-153 GACTGAGG(305)  DNA Index-153 GACTGAGG(305)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACTGAGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACTGAGGATCTCGTATGCCGTCTTCT
PCR-Free Index-153 PCR-Free Index-153
GCTTG(306)  GCTTG (306)
DNA Index-154 GTGTGTTA(307)  DNA Index-154 GTGTGTTA(307)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGTGTTAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGTGTTAATCTCGTATGCCGTCTTCT
PCR-Free Index-154 PCR-Free Index-154
GCTTG(308)  GCTTG (308)
DNA Index-155 CTCGTCCG(309)  DNA Index-155 CTCGTCCG(309)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCGTCCGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCGTCCGATCTCGTATGCCGTCTTCT
PCR-Free Index-155 PCR-Free Index-155
GCTTG(310)  GCTTG (310)
DNA Index-156 TGGAGAGG(311)  DNA Index-156 TGGAGAGG(311)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGGAGAGGATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGGAGAGGATCTCGTATGCCGTCTTCT
PCR-Free Index-156 PCR-Free Index-156
GCTTG(312)  GCTTG (312)
DNA Index-157 TGGAATTC(313)  DNA Index-157 TGGAATTC(313)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGGAATTCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGGAATTCATCTCGTATGCCGTCTTCT
PCR-Free Index-157 PCR-Free Index-157
GCTTG(314) DNA Index-158 TTGGCGCC(315) GCTTG (314) DNA Index-158 TTGGCGCC(315)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGGCGCCATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGGCGCCATCTCGTATGCCGTCTTCT
PCR-Free Index-158 PCR-Free Index-158
GCTTG(316)  GCTTG (316)
DNA Index-159 GCCTTAAT(317)  DNA Index-159 GCCTTAAT(317)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCTTAATATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCTTAATATCTCGTATGCCGTCTTCT
PCR-Free Index-159 PCR-Free Index-159
GCTTG(318)  GCTTG (318)
DNA Index-160 AAGCGATT(319)  DNA Index-160 AAGCGATT(319)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGCGATTATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGCGATTATCTCGTATGCCGTCTTCT
PCR-Free Index-160 PCR-Free Index-160
GCTTG(320)  GCTTG (320)
DNA Index-161 AACCGCAA(321)  DNA Index-161 AACCGCAA (321)
5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAACCGCAAATCTCGTATGCCGTCTTCT 5-Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACAACCGCAAATCTCGTATGCCGTCTTCT
PCR-Free Index-161 PCR-Free Index-161
GCTTG(322)  GCTTG (322)
利用上述根据本发明实施例的寡核苷酸， 能够有效地将 DNA 标签引入到样品的 DNA或其等同物中， 由此能够构建具有 DNA标签的 DNA标签文库。 另外， 发明人惊 奇地发现， 当针对相同的样品， 釆用具有不同标签的寡核苷酸构建含有各种 DNA标签 的 DNA标签文库时， 所得到的测序数据结果的稳定性和可重复性非常好。 根据本发明 的实施例， 当釆用 pearson系数进行数据分析时， 利用 DNA Indexl-161所构建的人全 血样本 DNA标签文库均表现出了至少 0.99的相关性。 关于 pearson系数具体算法的细 节可以参见相关文献， 例如： t Hoen, P. A., Y. Ariyurek, et al. (2008). "Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five micro array platforms." Nucleic Acids Res 36(21): el41 , 通过 参照将其全文并入本文。 重复性越高， 则其 pearson系数越接近 1。  With the above oligonucleotide according to an embodiment of the present invention, 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. In addition, 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. According to an embodiment of the present invention, 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. Ariyurek, et al. (2008). "Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over Five micro array platforms." Nucleic Acids Res 36(21): el41, which is incorporated herein by reference in its entirety. The higher the repeatability, the closer the pearson coefficient is to 1.
根据本发明的又一方面， 本发明提供了一种制备 DNA标签文库的方法。 根据本发 明的实施例， 其包括： 提供 DNA模板， 所述 DNA模板具有两条寡核苷酸链； 在所述 DNA 模板的两条寡核苷酸链的 3 '末端分别添加碱基 A； 在所述 DN A模板的两端分别连接含有 选自上述根据本发明实施例的一组分离的 DNA标签的一种的接头， 以便获得连接产物； 以及分离回收所述连接产物， 所述连接产物构成所述 DNA标签文库。 利用根据本发明 实施例的构建 DNA标签文库的方法， 能够有效地将根据本发明实施例的 DNA标签引入 到针对样品 DNA所构建的 DNA标签文库中。 从而可以通过对 DNA标签文库进行测序， 获得样品 DNA的序列信息以及 DNA标签的信息，从而能够对样品 DNA的来源进行区分。 另外， 发明人惊奇地发现， 当针对相同的样品， 基于上述方法， 釆用具有不同标签的寡 核苷酸构建含有各种 DNA标签的 DNA标签文库时， 所得到的测序数据结果的稳定性和 可重复性非常好。  According to still another aspect of the present invention, 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. With the method of constructing a DNA tag library according to an embodiment of the present invention, a DNA tag according to an embodiment of the present invention can be efficiently introduced into a DNA tag library constructed for sample DNA. Thus, 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. In addition, 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.
进一步， 本发明还提供了一种 DNA 标签文库， 其是由根据本发明实施例的构建 DNA标签文库的方法所获得的。  Further, 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.
根据本发明的又一方面， 本发明还提供了一种确定 DNA样品序列信息的方法。 根 据本发明的实施例， 其包括： 根据本发明实施例的构建 DNA标签文库的方法构建所述 DNA样品的 DNA标签文库； 以及对所述 DNA标签文库进行测序， 以便确定所述 DNA样 品的序列信息。 基于该方法， 能够有效地获得 DNA标签文库中 DNA样品的序列信息以 及 DNA标签的序列信息， 从而能够对 DNA样品的来源进行区分。 另外， 发明人惊奇地 发现， 利用根据本发明实施例的方法确定 DNA样品序列信息， 能够有效地减少数据产 出偏向性的问题， 并且能够精确地对多种 DNA标签文库进行区分。  According to still another aspect of the present invention, the present invention also provides a method of determining DNA sample sequence information. According to an embodiment of the present invention, 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. Based on this method, 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. Further, 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.
根据本发明的再一方面，本发明还提供了一种确定多种 DNA样品序列信息的方法。 根据本发明的实施例， 其包括以下步骤： 针对所述多种样品的每一种， 分别独立地根据 本发明实施例的构建 DNA标签文库的方法， 建立所述 DNA样品的 DNA标签文库， 其 中， 不同的 DNA样品釆用相互不同并且已知序列的 DNA标签， 其中所述多种为 2-161 种； 将所述多种样品的 DNA标签文库进行组合， 以便获得 DNA标签文库混合物； 利 用 Solexa测序技术， 对所述 DNA标签文库混合物进行测序， 以获得所述 DNA样品的 序列信息以及所述标签的序列信息； 以及基于所述标签的序列信息对所述 DNA样品的 序列信息进行分类， 以便确定所述多种样品的 DNA序列信息。 由此， 根据本发明实施 例的该方法， 可以充分利用高通量的测序技术， 例如利用 Solexa测序技术， 同时对多 种样品的 DNA标签文库进行测序， 从而提高 DNA标签文库测序的效率和通量， 同时 可以提高确定多种 DNA样品序列信息的效率。 According to still another aspect of the present invention, the present invention also provides a method of determining sequence information of a plurality of DNA samples. According to an embodiment of the present invention, 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. Thus, 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.
根据本发明的再一方面， 还提供了一种用于构建 DNA标签文库的试剂盒， 根据本 发明的实施例， 该试剂盒包括： 161种分离的寡核苷酸， 所述分离的寡核苷酸具有第一 链和第二链， 所述第一链由 SEQ ID NO: 323所示的核苷酸构成， 所述第二链由 SEQ ID NO: ( 2N ) 所示的核苷酸构成， 其中 N=l-161的整数， 其中， 所述 161种分离的寡核 苷酸分别设置在不同的容器中。 由此， 利用该试剂盒， 能够方便地将根据本发明实施例 的 DNA标签引入到构建的 DNA标签文库中。  According to still another aspect of the present invention, there is also provided a kit for constructing a DNA tag library, comprising: 161 isolated oligonucleotides, said isolated oligonucleotide, according to an embodiment of the present invention The nucleotide has a first strand consisting of the nucleotide set forth in SEQ ID NO: 323 and a second strand consisting of the nucleotide set forth in SEQ ID NO: (2N) Wherein N = an integer of 1 - 161, wherein the 161 isolated oligonucleotides are respectively disposed in different containers. Thus, with the kit, a DNA tag according to an embodiment of the present invention can be conveniently introduced into a constructed DNA tag library.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。  The additional aspects and advantages of the invention will be set forth in part in the description which follows.
附图说明 DRAWINGS
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：  The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from
图 1： 显示了 Illumina公司提供的 DNA标签文库构建方法的流程示意图； 图 2: 显示了根据本发明实施例的 DNA标签文库构建方法的流程示意图； 图 3: 显示了根据本发明实施例的构建的 44个 DNA标签文库的电泳结果。  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.
发明详细描述 Detailed description of the invention
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相 同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。  The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative only and not to limit the invention.
需要说明的是， 术语 "第一" 、 "第二" 仅用于描述目的， 而不能理解为指示或暗 示相对重要性或者隐含指明所指示的技术特征的数量。 由此， 限定有 "第一"、 "第二" 的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中， 除非另有说明， "多个" 的含义是两个或两个以上。  It should be noted that the terms "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.
DNA标签  DNA label
根据本申请的一个方面， 本发明提出了一些分离的 DNA标签。 根据本发明的实施 例， 这些分离的 DNA标签分别由 SEQ ID NO: ( 2N- 1 )所示的核苷酸序列构成， 其中 N=l-161的任意整数。 在本说明书中， 这些 DNA标签分别被命名为 DNA Index-N, 其 中 N=l-161的任意整数， 其序列如前面表 1所示， 在此不再赘述。  According to one aspect of the present application, the present invention proposes a number of isolated DNA tags. According to an embodiment of the present invention, these isolated DNA tags are each composed of the nucleotide sequence shown by SEQ ID NO: (2N-1), wherein N = any integer of 1-161. In the present specification, these DNA tags are respectively named DNA Index-N, wherein N=l-161 is an arbitrary integer, and the sequence thereof is as shown in Table 1 above, and will not be described herein.
在本发明中所使用术语 "DNA" 可以是任何包含脱氧核糖核苷酸的聚合物， 包括 但不限于经过修饰的或者未经修饰的 DNA。 利用根据本发明实施例的 DNA标签，通过 将 DNA标签与样品的 DNA或其等同物相连， 得到具有标签的 DNA标签文库， 通过对 DNA标签文库进行测序， 可以获得样品 DNA的序列以及标签的序列， 进而基于标签的 序列可以精确地表征 DNA的样品来源。 由此， 利用上述 DNA标签， 可以同时构建多 种样品的 DNA标签文库， 从而可以通过将来源于不同样品的 DNA标签文库进行混合， 同时进行测序， 基于 DNA标签对样品的 DNA序列进行分类， 获得多种样品的 DNA的 序列信息。 从而可以充分利用高通量的测序技术， 例如利用 Solexa测序技术， 同时对 多种样品的 DNA进行测序， 从而提高了通过高通量测序技术的效率和通量， 降低了确 定 DNA样品序列信息的成本。 这里所使用的表述方式 "DNA标签与样品的 DNA或其 等同物相连" 应做广义理解， 其包括 DNA标签可以与样品的 DNA直接相连， 以构建 DNA标签文库，也可以与和样品的 DNA具有相同序列的核酸（例如可以是相应的 RNA 序列或 cDNA序列， 其与 DNA具有相同的序列）相连。 The term "DNA" as used in the present invention may be any polymer comprising deoxyribonucleotides including, but not limited to, modified or unmodified DNA. Using 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. Thus, by using the above DNA tag, 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. Sequence information of DNA from a variety of samples. This allows for the full use of high-throughput sequencing technologies, such as the use of Solexa sequencing technology to simultaneously sequence DNA from multiple samples, thereby increasing the efficiency and throughput of high-throughput sequencing technologies and reducing the determination of DNA sample sequence information. cost. The expression "DNA tag attached to the DNA of the sample or its equivalent" as used herein 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.
本申请的发明人发现： 在本发明中， 为了设计有效的 DNA标签， 首先需要考虑标 签序列之间的可识别性和识别率的问题。 其次，在标签混合量少于 12个样品的情况下， 必须考虑到混合后的标签上的每个碱基位点的 GT含量。 因为 Solexa测序过程中，碱基 G和 T的激发荧光一样， 碱基 A和 C的激发光是一样的， 因此必须考虑碱基 "GT" 含 量与碱基 "AC" 含量的 "平衡" ， 最适碱基 "GT" 含量为 50% , 能保证标签识别率最 高和错误率最低。 最后， 还要考虑数据产出的可重复性和准确性， 即为了实现能够有效 构建 DNA标签文库并进行测序， 所构建的一组 DNA标签需要能够保证结果可靠， 可 重复性高， 也就是针对同样的 DNA样品， 可以保证利用该组 DNA标签中的不同标签 构建的 DNA标签文库， 能够获得一致的测序结果， 因而可以确保实验结果可靠且重复 性高。 另外， 还需要同时避免标签序列出现 3或 3 个以上连续的碱基的出现， 因为 3 个或 3个以上连续的碱基会增加序列在合成过程中或测序过程中的错误率，同时也要尽 量避免 DNA标签接头自身形成发夹结构。  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. 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.
为此， 本申请的发明人进行了大量的筛选工作， 并且选定了根据本发明实施例的一 组分离的 DNA标签， 其分别由 SEQ ID NO: ( 2N-1 ) 所示的核苷酸序列构成， 其中 N=l-161的任意整数。 其序列如前面表 1所示， 不再赘述。 另外， 发明人发现这些标签 之间的差异至少有 4个碱基， 即至少 4个碱基序列不同， 并且当标签的 8个碱基中的任 意 1个碱基出现测序错误或合成错误， 都不影响到标签的最终识别。这些标签可以应用 于任何 DNA标签文库的构建。 目前尚未有关于这些标签应用于 DNA样品测序的文库 构建并通过 Solexa测序的报道。  To this end, 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 constitutes, wherein N = any integer of l-161. The sequence is as shown in Table 1 above and will not be described again. In addition, 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.
根据本发明的一些实施例， 所釆用的 DNA标签为长度是 8bp的核酸序列， 并且所述 标签之间的差异在 4个碱基以上， 所述一组 DNA标签由如下组成： 161个 DNA标签或与 之相差 1个碱基的 DNA标签中的至少 10个， 或至少 20个， 或至少 30个， 或至少 40个， 至 少 50个， 或至少 60个， 或至少 70个， 或至少 80个， 或 90个， 或至少 100个， 或至少 110 个， 或至少 120个， 或至少 130个， 或至少 140个， 或至少 150个， 或全部 161个。 具体地， 根据本发明的实施例， 所述一组 DNA标签优选地至少包括表 1所示的 161个 DNA标签的 DNA Index 1 ~ DNA Index 10, 或 DNA Indexl l ~ DNA Index20, 或 DNA Index21 ~ DNA Index30 , 或 DNA Index31 - DNA Index40 , 或 DNA Index ~ DNA Index50 , 或 DNA Index51 ~ DNA Index60 , 或 DNA Index61 ~ DNA Index70 , 或 DNA Index71 ~ DNA Index80, 或 DNA Index81 ~ DNA Index90, 或 DNA Index91 ~ DNA IndexlOO , 或 DNA Index 101 - DNA Index 110 , 或 DNA Index 111 - DNA Index 120, 或 DNA Index 121 ~ DNA Index 130,或 DNA Index l ~ DNA Index 140 ,或 DNA Indexl41 ~ DNA Index 150 ,或 DNA Index 151 - DNA Indexl61 , 或者他们任何两个或多个的组合。在本发明的一些具体示例 中， 所述相差 1个碱基包括对表 1所示 161个标签的序列中 1个碱基的取代、 添加或缺失。  According to some embodiments of the invention, 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. Specifically, according to an embodiment of the present invention, 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. In some specific examples of the invention, the 1 base difference comprises a substitution, addition or deletion of 1 base in the sequence of 161 tags shown in Table 1.
根据本发明的实施例， 本发明还提供了将根据本发明实施例的标签用于 DNA标签 文库构建并测序的用途， 其中 DNA标签文库的 DNA标签接头包含根据本发明实施例的 DNA标签， 从而构成各自相对应的 DNA标签接头。 根据该用途的实施例， DNA标签插 入 DNA PCR-Free标签接头中， 或通过或不通过连接子连接在 DNA接头的 3'末端， 优选 地插入 DNA PCR-Free标签接头中。 根据具体的示例， 连接子是 1-6个核苷酸序列， 优选 地 1-3个核苷酸序列。  According to an embodiment of the present invention, 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. According to an embodiment of this use, 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. According to a specific example, the linker is a 1-6 nucleotide sequence, preferably a 1-3 nucleotide sequence.
寡核苷酸以及构建 DNA标签文库  Oligonucleotides and construction of DNA tag libraries
根据本发明的又一方面， 本发明提供了一组分离的寡核苷酸， 其可以用于将前面所 描述的 DNA标签引入到样品的 DNA中， 进而构建 DNA标签文库。 根据本发明的实施例， 本发明提供了一组分离的寡核苷酸， 该组分离的寡核苷酸中的每一种均具有粘性末端 T, 并且这些分离的寡核苷酸具有第一链和第二链， 粘性末端 T形成在每一种寡核苷酸 的第一链上。 其中， 根据本发明的实施例， 第一链由 SEQ ID NO: 323所示的核苷酸构 成， 第二链分别由 SEQ ID NO: ( 2N ) 所示的核苷酸构成， 其中 N=l-161的任意整数。 本领域技术人员能够理解，可以通过分别将构成相应寡核苷酸的第一链与第二链进行退 火处理， 而形成相应的寡核苷酸。 根据本发明的实施例， 上述寡核苷酸分别具有如前所 述的根据本发明实施例的 DNA标签， 并且这些寡核苷酸具有粘性末端， 因而， 可以通 过连接反应， 将相应的 DNA标签引入到样品的 DNA或其等同物中。 具体地， 这些寡核 苷酸的序列如前面表 1所示， 在此不再赘述。 According to yet another aspect of the invention, 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. According to an embodiment of the invention, 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. Wherein, according to an embodiment of the present invention, the first strand is composed of the nucleotide represented by SEQ ID NO: 323 The second strand is composed of the nucleotides represented by SEQ ID NO: (2N), respectively, wherein N = any integer of 1-161. Those skilled in the art will appreciate that the corresponding oligonucleotides can be formed by annealing the first strand and the second strand constituting the corresponding oligonucleotide, respectively. According to an embodiment of the present invention, 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.
发明人发现， 根据本发明的实施例所提供的寡核苷酸序列 （ DNA PCR-Free标签接 头） 具有较高的稳定性。 该发现主要是根据本发明的一些实施例， 通过 Lasergene软件 ( http://www.dnastar.com/ ) 分析测试这些寡核苷酸序列的结构稳定性得来的。 使用 Lasergene的 PrimerSelect软件， 通过分析两条序列之间形成的能量值可以判断双链体之 间的亲和力参数， 从而预测 DNA PCR-Free标签接头形成的最稳定二聚体结构（ the most stable dimer overran )及能量值， 其中， 能量值（ kcal/mol ) 的绝对值越大， 表示双链体 的结果越稳定。 以下是分别对前面表 1所示的 161个 DNA PCR-Free标签接头进行上述的 结构稳定性和亲和力分析的结果， 结果表明， 这些 DNA PCR-Free标签接头形成的 "Y 型" 结构非常稳定。  The inventors have found that the oligonucleotide sequence (DNA PCR-Free tag junction) provided according to an embodiment of the present invention has high stability. This finding was primarily based on the analysis of the structural stability of these oligonucleotide sequences by Lasergene software (http://www.dnastar.com/) in accordance with some embodiments of the present invention. Using Lasergene's PrimerSelect software, 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.
下面提供了根据本发明实施例的 DNA PCR-Free 标签接头的二级结构以及最稳定 的二聚体结构 （ The most stable dimer overall ) —— "γ型" 结构及其能量值。  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.
DNA PCR-Free index 1 接头  DNA PCR-Free index 1 connector
The The
Figure imgf000017_0001
Figure imgf000017_0001
3 f TCTAGCC TC CGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3 f TCTAGCC TC CGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index2 接头
Figure imgf000017_0002
DNA PCR-Free index2 connector
Figure imgf000017_0002
DNA PCR-Free index3 接头
Figure imgf000017_0003
DNA PCR-Free index3 connector
Figure imgf000017_0003
DNA PCR-Free index4 接头
Figure imgf000017_0004
DNA PCR-Free index4 connector
Figure imgf000017_0004
DNA PCR-Free index5 接头 DNA PCR-Free index5 connector
The most st— able dii er cver l I： 12 bp, -22 . 8 kcs丄 ½ 1
Figure imgf000017_0005
The most st- able dii er cver l I: 12 bp, -22 . 8 kcs丄1⁄2 1
Figure imgf000017_0005
DNA PCR-Free index6 接头  DNA PCR-Free index6 connector
The most stable dlirser o v erall： 12 fop , -22 . 8 kcal/mol  The most stable dlirser o v erall: 12 fop , -22 . 8 kcal/mol
5 ' GATCGGAAGAGCACACGTCTGAAGTCCAG; TCACCAACAGGTATCTCGTATGCCGTCTTCTGCTTG5 ' GATCGGAAGAGCACACGTCTGAAGTCCAG ; TCACCAACAGGTATCTCGTATGCCGTCTTCTGCTTG
Figure imgf000017_0006
Figure imgf000017_0006
DNA PCR-Free index7 接头 The ： mo st stable dimer over ll： 12 bp, -22. δ kcal/mol DNA PCR-Free index7 connector The : mo st stable dimer over ll: 12 bp, -22. δ kcal/mol
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTCAAGGTATCTCGTATGCCGTCTTCTGCTTG 3 ' 5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTCAAGGTATCTCGTATGCCGTCTTCTGCTTG 3 '
3 T TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 51 3 T TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 1
DNA PCR-Free index8 接头 DNA PCR-Free index8 connector
The most stable dirr.er overall: 12 bp, -22.8 kcal/mol  The most stable dirr.er overall: 12 bp, -22.8 kcal/mol
3，■ TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3,■ TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index9 接头  DNA PCR-Free index9 connector
The ost, stable d.im.er overall： 12 bp, -22 ' 8 kcal 丄  The ost, stable d.im.er overall: 12 bp, -22 ' 8 kcal 丄
5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCACCACGTAGTATCTCGTATGCCGTCTTCTGCTTG 3T 5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCACCACGTAGTATCTCGTATGCCGTCTTCTGCTTG 3 T
5'  5'
DNA PCR-Free index 10 接头 DNA PCR-Free index 10 connector
The most stable dimer overall： 12 bp , -22.8 kcal /TJIOI  The most stable dimer overall: 12 bp , -22.8 kcal /TJIOI
31 TCTAGCGT CTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5， 3 1 TCTAGCGT CTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5,
DNA PCR-Free index 11 接头 DNA PCR-Free index 11 connector
The most stable dinner overall： 12 bpf -22. Θ kcal/mol The most stable dinner overall: 12 bp f -22. Θ kcal/mol
5 ' GA CGGAAGAGCACACGTCTGAAC CCAGTCACTACCTTCTATCTCGTATGCCGTCTTCTGCTTG 31 5 ' GA CGGAAGAGCACACGTCTGAAC CCAGTCACTACCTTCTATCTCGTATGCCGTCTTCTGCTTG 3 1
^CGGCATAGTAA 5f ^CGGCATAGTAA 5 f
DNA PCR-Free indexl2 接头  DNA PCR-Free indexl2 connector
The most s ble d liner overall： 上 2 b , -22. Θ kcal/mol  The most s ble d liner overall: upper 2 b , -22. Θ kcal/mol
5 ' GAT GGAAGAGCACACG  5 ' GAT GGAAGAGCACACG
CGGCATAGTAA 5 f CGGCATAGTAA 5 f
DNA PCR-Free index 13 接头 DNA PCR-Free index 13 connector
The most s able diir- r overall： 12 bp f -22. S kcal/mol The most s able diir- r overall: 12 bp f -22. S kcal/mol
DNA PCR-Free indexl4 接头 DNA PCR-Free indexl4 connector
The most stable dimer ove.rall： .12 b r -22.8 kcal/ir:o丄 3 ' TCTAGCCTTGTCGCAGCACATGGCTTTCTCACAT TAGAGCCACCAGCGGCATAGTAA The most stable dimer ove.rall: .12 b r -22.8 kcal/ir:o丄3 ' TCTAGCCTTGTCGCAGCACATGGCTTTCTCACAT TAGAGCCACCAGCGGCATAGTAA
DNA PCR-Free index 15 接头 DNA PCR-Free index 15 connector
The mos stable diiiter ov r ll； 12 bp, -22.8 kcal/irtol  The mos stable diiiter ov r ll; 12 bp, -22.8 kcal/irtol
3 ' TC AGCCTTCTCGCAGCACA CCCTT CTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3 ' TC AGCCTTCTCGCAGCACA CCCTT CTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index 16 接头  DNA PCR-Free index 16 connector
The most stable dii^er overall： 12 bp, -22.8 kcal/mol  The most stable dii^er overall: 12 bp, -22.8 kcal/mol
DNA PCR-Free indexl7 接头 The most stable dim r overall： 12 b f -22. δ kcal /mo I DNA PCR-Free indexl7 connector The most stable dim r overall: 12 b f -22. δ kcal /mo I
J GATCGGAAGAGCACAC  J GATCGGAAGAGCACAC
3 f TCTAGCCTTC CGCAG 3 f TCTAGCCTTC CGCAG
DNA PCR-Free index 18 接头  DNA PCR-Free index 18 connector
The Ffios t stable dime r over ll： 12 bp, - 22. S kcal /niol  The Ffios t stable dime r over ll: 12 bp, - 22. S kcal /niol
DNA PCR-Free index 19 接头 DNA PCR-Free index 19 connector
The most stable dimer overall： 12 fopf -22.8 The most stable dimer overall: 12 fop f -22.8
T GATCGGAAGAGCACACGTCTGAACTCCAGTCACC AGTT7  T GATCGGAAGAGCACACGTCTGAACTCCAGTCACC AGTT7
3 f TCT¾GCCT C CGCAGCACATCCCTTTCTGACATCTAGAGCCACCAGCGGCA AGTAA 5 ' 3 f TCT3⁄4GCCT C CGCAGCACATCCCTTTCTGACATCTAGAGCCACCAGCGGCA AGTAA 5 '
DNA PCR-Free index20 接头 DNA PCR-Free index20 connector
The most stable dimer overall： 12 bp -22.8 kcal/inol  The most stable dimer overall: 12 bp -22.8 kcal/inol
5  5
； CATSGTAA 5 '  ; CATSGTAA 5 '
DNA PCR-Free index21 接头 DNA PCR-Free index21 connector
The most, stable dimer overall： 12 -22.8 kcal/mo丄  The most, stable dimer overall: 12 -22.8 kcal/mo丄
51 5 1
DNA PCR-Free index22 接头 DNA PCR-Free index22 connector
The most s table airier overall： 12 bp - 8 kcaI ϊΓ-ο!  The most s table airier overall: 12 bp - 8 kcaI ϊΓ-ο!
5 i L.TCi .GA A CTCGTA GCCGTC TCTGCTTG 5 i L .TCi .GA A CTCGTA GCCGTC TCTGCTTG
5CGGCA AGTAA 5 ' 5CGGCA AGTAA 5 '
DNA PCR-Free index23 接头 DNA PCR-Free index23 connector
The most stable dimer overall： 丄 2 bp, —22, 8 kcal/ ol  The most stable dimer overall: 丄 2 bp, —22, 8 kcal/ ol
51 GA CGGAAGAGCACACGTCTGAACTCCAGTCACTATGCCATATCTCGTATGCCGTCTTCTGCTTG5 1 GA CGGAAGAGCACACGTCTGAACTCCAGTCACTATGCCATATCTCGTATGCCGTCTTCTGCTTG
3， TCTAGCCTTCTCGCAG ACATCCCTTTCTCACATC AGAGCCACCAGCGGCATAGTAA 5， 3, TCTAGCCTTCTCGCAG ACATCCCTTTCTCACATC AGAGCCACCAGCGGCATAGTAA 5,
DNA PCR-Free index24 接头  DNA PCR-Free index24 connector
The most stable diir-er overall： 12 b F -22.8 kcal/irto丄 The most stable diir-er overall: 12 b F -22.8 kcal/irto丄
5 f GA CGGAAGAG 5 f GA CGGAAGAG
3， C AGC C T TC T C GC AG  3, C AGC C T TC T C GC AG
DNA PCR-Free index25 接头 DNA PCR-Free index25 connector
The mo t stable dimer overall： 12 bp, -22.8 kcal /mo I  The mo t stable dimer overall: 12 bp, -22.8 kcal /mo I
31 C AGCC C CGCAGCACA C C C C：ACA C A AGCGACCAGCGGCA AG AA 5，3 1 C AGCC C CGCAGCACA CCCC: ACA CA AGCGACCAGCGGCA AG AA 5,
DNA PCR-Free index26 接头 DNA PCR-Free index26 connector
The most stable dimer overall： 12 fop , -22.8 kcal /mo I  The most stable dimer overall: 12 fop , -22.8 kcal /mo I
3 τ 3 τ
DNA PCR-Free index27 接头 The most st ble dimei: ov rall： 丄 2 bp, -22.8 kcal /mol DNA PCR-Free index27 connector The most st ble dimei: ov rall: 丄2 bp, -22.8 kcal /mol
5 f 5 f
DNA PCR-Free index28 接头 DNA PCR-Free index28 connector
The most st b le dir er overall： 12 bp, - 22.. S kc l/'r ol  The most st b le dir er overall: 12 bp, - 22.. S kc l/'r ol
DNA PCR-Free index29 接头 DNA PCR-Free index29 connector
most s able dimer overall： I 2 -22.8 kcs丄 /rrtoi  Most s able dimer overall: I 2 -22.8 kcs丄 /rrtoi
I M M I f  I M M I f
,GTAA 5，  , GTAA 5,
DNA PCR-Free index30 接头  DNA PCR-Free index30 connector
The iTiGst stable dimer overall: 12 bp, -22. S kcal moi  The iTiGst stable dimer overall: 12 bp, -22. S kcal moi
5，' GATCGGAAGAGCACACGTC GAACTCCAGTCACGGATA GGATCTCGTATGCCGTCTTCTGC TG 3 5,' GATCGGAAGAGCACACGTC GAACTCCAGTCACGGATA GGATCTCGTATGCCGTCTTCTGC TG 3
31 TCTAGCCTTCTCGCAGC^CATCCCTTTCTCACATCTAGAGCC^C^^ 5 ' 3 1 TCTAGCCTTCTCGCAGC^CATCCCTTTCTCACATCTAGAGCC^C^^ 5 '
DNA PCR-Free index31 接头  DNA PCR-Free index31 connector
The most stable dlmer overall： 12 bp, -22.8 kcal irtol  The most stable dlmer overall: 12 bp, -22.8 kcal irtol
5 T GATCGGAAGAGCACAC 5 T GATCGGAAGAGCACAC
31 TCTAGCCTTCTCGCAGC 3 1 TCTAGCCTTCTCGCAGC
DNA PCR-Free index32 接头  DNA PCR-Free index32 connector
The most stable d. inter oversl丄： 12 , -22.8 k al/nol  The most stable d. inter oversl丄: 12 , -22.8 k al/nol
5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCACAAGATCGGATCTCGTATCCCGTCTTCTGC TG 3 ' 5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCACAAGATCGGATCTCGTATCCCGTCTTCTGC TG 3 '
3 T TCTAGCCTTCTCGCAGCACA CCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3 T TCTAGCCTTCTCGCAGCACA CCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index33 接头  DNA PCR-Free index33 connector
The most st ble dimer overall: i2 b r -22.8 kcal /mol The most st ble dimer overall: i2 b r -22.8 kcal /mol
3 f TCTAGCC TCTCGCAGCACATCCC TTCTCACATC AGAGv CACCAGCGGCA AGTAA 5 ' 3 f TCTAGCC TCTCGCAGCACATCCC TTCTCACATC AGAGv CACCAGCGGCA AGTAA 5 '
DNA PCR-Free index34 接头  DNA PCR-Free index34 connector
The most s able di er overall： 12 b , -22. S kcal ΙΪΪΟΙ  The most s able di er overall: 12 b , -22. S kcal ΙΪΪΟΙ
5 ' GAT GGAAGAGCACACGTCTGAACTCCAG GACCTAAGTCGATCTCGTATGCCGTCTT TGCTTG 3 ' 5 ' GAT GGAAGAGCACACGTCTGAACTCCAG GACCTAAGTCGATCTCGTATGCCGTCTT TGCTTG 3 '
31 TCTAGCCTTCTCGCAGCACATCCCTT TCACATCTAGAGCCACCAGCGGCATAGTAA 51 3 1 TCTAGCCTTCTCGCAGCACATCCCTT TCACATCTAGAGCCACCAGCGGCATAGTAA 5 1
DNA PCR-Free index35 接头 DNA PCR-Free index35 connector
The most stable dimer overall； 12 fop, -22.8 kcal/moi  The most stable dimer overall; 12 fop, -22.8 kcal/moi
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATTCGCGATCTCGTATGCCGTCTTCTGCTTG 3 ' 5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATTCGCGATCTCGTATGCCGTCTTCTGCTTG 3 '
； CGGCATAGTAA 5， ; CGGCATAGTAA 5,
DNA PCR-Free index36 接头  DNA PCR-Free index36 connector
The most s able d丄 inei: overall： 12 bp , -22.8  The most s able d丄 inei: overall: 12 bp , -22.8
GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAAGCACGATCTCGTATGCCGTCTTCTGCTTG 3 '  GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAAGCACGATCTCGTATGCCGTCTTCTGCTTG 3 '
LGCGGCA AGTAA  LGCGGCA AGTAA
DNA PCR-Free index37 接头 NCeede DA PRFr inx47- CGGCATAGDNA PCR-Free index37 connector NCeede DA PRFr inx47- CGGCATAG
Figure imgf000021_0001
Figure imgf000021_0001
, ... O, ... O
?NCeede5 DA PRFr inx7— ? NCeede5 DA PRFr inx7—
CCACAGC^GCATACTAA!4一 p ,vTheost: szable dimer overall 12 b22.8 kcaili - . CCACAGC^GCATACTAA!4_p,vTheost: szable dimer overall 12 b22.8 kcaili - .
?NCeede56 DA PRFr inx- ACCAGCGGCATAG AA!I】  ? NCeede56 DA PRFr inx- ACCAGCGGCATAG AA!I]
一 One
¾/The most stale di overall: 12 b kcalol- " 3⁄4/The most stale di overall: 12 b kcalol- "
NCeede55 DA PRFr inx- . NCeede55 DA PRFr inx- .
The .  The .
--
The
Figure imgf000022_0001
The
Figure imgf000022_0001
p,/T stable cvera： b kcalmoi . p, /T stable cvera: b kcalmoi .
NCeede5 DA PRFr inx1- p、/The stable dimer overall： 12 b kcalmoj. . NCeede5 DA PRFr inx1- p, /The stable dimer overall: 12 b kcalmoj.
?NCeede50 DA PRFr inx- 一 ? NCeede50 DA PRFr inx- one
p,The most; stable dinier overall: b kcai - NCeede9 DA PRFr inx4- p/ The most; stable dier overall.: b kcai3yol- NCeede8 DA PRFr inx4- The most stable d imer overal 1： 12 bp, -22.8 p,The most; stable dinier overall: b kcai - NCeede9 DA PRFr inx4- p/ The most; stable dier overall.: b kcai3yol- NCeede8 DA PRFr inx4- The most stable d imer overal 1: 12 bp, -22.8
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAAT ACCATCTCGTA GCCGTCT CTGCT G 3 ' 5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAAT ACCATCTCGTA GCCGTCT CTGCT G 3 '
31 CTAGCCTTC CGCAGCACATCCCTTTCTCACA G AGAGCCACCAGCGGCA.TAGTAA. 5 ' 3 1 CTAGCCTTC CGCAGCACATCCCTTTCTCACA G AGAGCCACCAGCGGCA.TAGTAA. 5 '
DNA PCR-Free index58 接头  DNA PCR-Free index58 connector
The most stable dir^.er o e r 11； 12 bp, -22.8  The most stable dir^.er o e r 11; 12 bp, -22.8
5 " GATCGGAAGAGCACi  5 " GATCGGAAGAGCACi
DNA PCR-Free index59 接头 DNA PCR-Free index59 connector
The most st ble d lin overall； 12 fop, -22.8 kcsl/m il  The most st ble d lin overall; 12 fop, -22.8 kcsl/m il
5 ' GA'TCGGAAG^  5 ' GA'TCGGAAG^
LGCGGCATAGTAA 5 '  LGCGGCATAGTAA 5 '
DNA PCR-Free index60 接头  DNA PCR-Free index60 connector
■most .s abl dime r overall： 12 bp, -22. S kc l r^ol  ■most .s abl dime r overall: 12 bp, -22. S kc l r^ol
M M M M
TCTAG CTTCT GCi^ TCTAG CTTCT GCi^
DNA PCR-Free index61 接头  DNA PCR-Free index61 connector
The most .st ble :er overall： 12 bp, -22. S kcal κι.ο·!  The most .st ble :er overall: 12 bp, -22. S kcal κι.ο·!
5 ' GATCTG.AAGAGCACACGTCTGAAC CCAGTCACCTCTCGACA CTCG ATGCCGTCT CTGC TG 3  5 ' GATCTG.AAGAGCACACGTCTGAAC CCAGTCACCTCTCGACA CTCG ATGCCGTCT CTGC TG 3
DNA PCR-Free index62 接头 DNA PCR-Free index62 connector
The ost stable dimer overall： 12 bp , -22. B kcal /mol  The ost stable dimer overall: 12 bp , -22. B kcal /mol
3 T TCTAGCGTTCTCGCAGCACATCCGTTTCTGACATC AGAGCCACCAGCGGCA AGTAA 5 ' 3 T TCTAGCGTTCTCGCAGCACATCCGTTTCTGACATC AGAGCCACCAGCGGCA AGTAA 5 '
DNA PCR-Free index63 接头  DNA PCR-Free index63 connector
The mos stable dim ov rall： 12 b T -22.8 kcal / mol The mos stable dim ov rall: 12 b T -22.8 kcal / mol
5 ' GATCGGAAGAGCACACf  5 ' GATCGGAAGAGCACACf
M M  M M
DNA PCR-Free index64 接头 DNA PCR-Free index64 connector
The most stable imei: ov r ll： 12 b -22.8 kcal /mol  The most stable imei: ov r ll: 12 b -22.8 kcal /mol
5，  5,
DNA PCR-Free index65 接头 DNA PCR-Free index65 connector
The mo t stable dimer overall: 12 bp, -22.8 kcsl/rrtoi  The mo t stable dimer overall: 12 bp, -22.8 kcsl/rrtoi
5,  5,
JCCACCAGCGGCATAGTAA ，  JCCACCAGCGGCATAGTAA,
DNA PCR-Free index66 接头 DNA PCR-Free index66 connector
he ir-os t stable dimer overall： 12 bp, -22.8 kcal /l ol  He ir-os t stable dimer overall: 12 bp, -22.8 kcal /l ol
5 » GATCGGAAGAGCACACC  5 » GATCGGAAGAGCACACC
31 TCTAGCC TC CGCAG 3 1 TCTAGCC TC CGCAG
DNA PCR-Free index67 接头 The s able dimer overall： 丄 2 bp, -22.8 kcal/ir-ol DNA PCR-Free index67 connector The s able dimer overall: 丄2 bp, -22.8 kcal/ir-ol
5 T GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTACCATAATCTCGTATGCCGTCTTCTGCTTG 3 ' 5 T GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTACCATAATCTCGTATGCCGTCTTCTGCTTG 3 '
.GCGGCA AGTAA 51 .GCGGCA AGTAA 5 1
DNA PCR-Free index68 接头  DNA PCR-Free index68 connector
The most st ble dimer over ll： 12 bp, —22.8 kca丄 /ίπο  The most st ble dimer over ll: 12 bp, —22.8 kca丄 /ίπο
5， GATCGGAAGAGCACACC  5, GATCGGAAGAGCACACC
3 T TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATC AGAGCCACCAGCGGCATAGTAA 5 ' 3 T TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATC AGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index69 接头  DNA PCR-Free index69 connector
The mos stable ov rall： 12 bp, -22.8 kcal /rrtol  The mos stable ov rall: 12 bp, -22.8 kcal /rrtol
3 ' TCTAGCCTTCTCGCAGCACATCCCTTT TCACAT TAGAGCCACCAGCGGCATAGTiA 5， 3 ' TCTAGCCTTCTCGCAGCACATCCCTTT TCACAT TAGAGCCACCAGCGGCATAGTiA 5,
DNA PCR-Free index70 接头  DNA PCR-Free index70 connector
The most stabl dimer overall： 12 bp, -22.8 kcal/ττιοϊ  The most stabl dimer overall: 12 bp, -22.8 kcal/ττιοϊ
DNA PCR-Free index71 接头 DNA PCR-Free index71 connector
The most stable dim r overall： 12 bp, -22 8 kcal/rn l  The most stable dim r overall: 12 bp, -22 8 kcal/rn l
GATCGGAAGAGCACACGTCTGAACTCCAG CAC ACTA GAATCTCGTATGCCCTCT  GATCGGAAGAGCACACGTCTGAACTCCAG CAC ACTA GAATCTCGTATGCCCTCT
DNA PCR-Free index72 接头 DNA PCR-Free index72 connector
The most s abl dimer overall： 上 2 bp f -22.8 kcal/mo丄 CACCAGCGGCATAGTAA 5 ' The most s abl dimer overall: upper 2 bp f -22.8 kcal/mo丄CACCAGCGGCATAGTAA 5 '
DNA PCR-Free index73 接头  DNA PCR-Free index73 connector
The most stable diiTier overall： 1.2 bp , -22.8 kcal /mo 1  The most stable diiTier overall: 1.2 bp , -22.8 kcal /mo 1
GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTGAGGAATCTCGTATGCCG CTTC GCTT 3  GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTGAGGAATCTCGTATGCCG CTTC GCTT 3
DNA PCR-Free index74 接头 DNA PCR-Free index74 connector
The most stable dimer overall； 丄 2 b f -22.8 kcal /mol The most stable dimer overall; 丄2 b f -22.8 kcal /mol
5， GA CGGAAGAGCACACGTCTGAACTCCAGTCACATCGCCGAATCTCGTATGCCG CTTCTGCT G 3 '  5, GA CGGAAGAGCACACGTCTGAACTCCAGTCACATCGCCGAATCTCGTATGCCG CTTCTGCT G 3 '
DNA PCR-Free index75 接头 DNA PCR-Free index75 connector
The most stable d iii r c-vera丄 1： 12 b r -22.8 kcal/mol CGGCATAGTAAThe most stable d iii r c-vera丄1: 12 b r -22.8 kcal/mol CGGCATAGTAA
DNA PCR-Free index76 接头 DNA PCR-Free index76 connector
The mo st. st 丄 e dime r ov ra l: 丄 2 bp -22.8 kcsl/mo丄  The mo st. st 丄 e dime r ov ra l: 丄 2 bp -22.8 kcsl/mo丄
.5， GA CGGAA-:  .5, GA CGGAA-:
： CAGCGGCATAGTAA  : CAGCGGCATAGTAA
DNA PCR-Free index77 接头 The most stable diir:er overall： 12 bp, -22. S kcal/mol DNA PCR-Free index77 connector The most stable diir:er overall: 12 bp, -22. S kcal/mol
5 ' GATCGGAAGAGi  5 ' GATCGGAAGAGi
DNA PCR-Free index78 接头 DNA PCR-Free index78 connector
The most s able di er overall： 12 fo T -22.3 kcal /ITEO! The most s able di er overall: 12 fo T -22.3 kcal /ITEO!
51 GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCTTGCAATCTCGTATGCCGTCTTCTGC TG 3 5 1 GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCTTGCAATCTCGTATGCCGTCTTCTGC TG 3
DNA PCR-Free index79 接头 DNA PCR-Free index79 connector
The most stable dii^er oversl丄： 12 bp, -22 8 kc-al./ 丄  The most stable dii^er oversl丄: 12 bp, -22 8 kc-al./ 丄
5， GATCGGAAGAGCACACGTCTGAACTCCAGTCACATACTCCAATCTCGTATGCCGTCTTCTGCTTG 3，  5, GATCGGAAGAGCACACGTCTGAACTCCAGTCACATACTCCAATCTCGTATGCCGTCTTCTGCTTG 3,
DNA PCR-Free index80 接头 DNA PCR-Free index80 connector
The o:::t stable dimer overall： 1 bp, -22.8 kcal /mo I  The o:::t stable dimer overall: 1 bp, -22.8 kcal /mo I
' GATCGGAAGAGCACACGTCTGAACTCCAG CACGTTCGACAATCTCGTATGCCGTCT CTGCT G 1 ' GATCGGAAGAGCACACGTCTGAACTCCAG CACGTTCGACAATCTCGTATGCCGTCT CTGCT G 1
DNA PCR-Free index81 接头 DNA PCR-Free index81 connector
The most, s able dimer overall: 12 b f -22.8 kcal/mol The most, s able dimer overall: 12 b f -22.8 kcal/mol
5 GATCGGAAGAGCACACGTCTGAAC  5 GATCGGAAGAGCACACGTCTGAAC
DNA PCR-Free index82 接头 DNA PCR-Free index82 connector
The most s able dirrt r overall： 12 bp, -22. S k al -ol  The most s able dirrt r overall: 12 bp, -22. S k al -ol
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACATGAAATCTCGTATGCCGTCTTC GCTTG 5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACATGAAATCTCGTATGCCGTCTTC GCTTG
31 TCTAGCCTTCTCGCAGCACATCCCTT CTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3 1 TCTAGCCTTCTCGCAGCACATCCCTT CTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index83 接头 DNA PCR-Free index83 connector
he mo t stable diriier overall： 1.2 b , -22, 8 kca丄 /irol  He mo t stable diriier overall: 1.2 b , -22, 8 kca丄 /irol
5 ' GATCGGAAGAGCACAC-GTCTGAACTCCAGTCACATGAGGAAATCTCGTATC^CGTCTTCTGCTTG 5 ' GATCGGAAGAGCACAC-GTCTGAACTCCAGTCACATGAGGAAATCTCGTATC^CGTCTTCTGCTTG
3 f TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3 f TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index84 接头  DNA PCR-Free index84 connector
The most stable d int r overall： 12 bp, -22..8 kcsl/ ol  The most stable d int r overall: 12 bp, -22..8 kcsl/ ol
5 ' GATCGGAAGAGCACACGTCTGAAC CCAGTCACTCCTCCAAA CTCGTATGCCGTCTTCTGCTTG  5 ' GATCGGAAGAGCACACGTCTGAAC CCAGTCACTCCTCCAAA CTCGTATGCCGTCTTCTGCTTG
DNA PCR-Free index85 接头 DNA PCR-Free index85 connector
The most st ble dimer cveral I： 12 bp, -22.8 k l /mol  The most st ble dimer cveral I: 12 bp, -22.8 k l /mol
LGCCACCAGCGGCA AGTAA LGCCACCAGCGGCA AGTAA
DNA PCR-Free index86 接头 DNA PCR-Free index86 connector
The mos s ble diir-er ov r ll： 12 bp, -22.8 kcs丄 / o丄  The mos s ble diir-er ov r ll: 12 bp, -22.8 kcs丄 / o丄
DNA PCR-Free index87 接头 DNA PCR-Free index87 connector
?NCeede9 DA PRFr inx7— ? NCeede9 DA PRFr inx7—
3 _3 _
p, The ms t stable dimer overall b · β p, The ms t stable dimer overall b · β
?NCeede93 DA PRFr inx- p/he most;tabl_e dimer overall2 b22 , 8 kcalmol 1-... NCeede93 DA PRFr inx- p/he most;tabl_e dimer overall2 b22 , 8 kcalmol 1-...
?NCeede9 DA PRFr inx2-
Figure imgf000026_0001
?NCeede9 DA PRFr inx2-
Figure imgf000026_0001
mc overall; 1222,8 kca3 - ?NCeede9 DA PRFr inx1— _ The most stabie di_er over . kcall.!-. " NCeede90 DA PRFr inx- Mc overall; 1222,8 kca3 - ?NCeede9 DA PRFr inx1— _ The most stabie di_er over . kcall.!-. " NCeede90 DA PRFr inx-
The most stable dirrter overall： 12 bp, -22. S k al /mo I The most stable dirrter overall: 12 bp, -22. S k al /mo I
31 TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATC AGAGCCACCAG GGCATAGTAA 5 ' 3 1 TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATC AGAGCCACCAG GGCATAGTAA 5 '
DNA PCR-Free index98 接头  DNA PCR-Free index98 connector
The most stable dimer overall： i 2 bp -22. S kcal oi  The most stable dimer overall: i 2 bp -22. S kcal oi
5 f GATCGGAAGAGCACACG 5 f GATCGGAAGAGCACACG
31 CTAGCCTTC CGCAG 3 1 CTAGCCTTC CGCAG
DNA PCR-Free index99 接头  DNA PCR-Free index99 connector
most stable dim r overall： 12 fop, -22 8 kcal /mol  Most stable dim r overall: 12 fop, -22 8 kcal /mol
GATCGCAAGAGCACACGTCTGAACTCCACTCACGAGCATTGATCTCGTATGCCGTCTTCTGC TCTAGCCTT TCGCAGCACATCv C TTCTCACATv AGAGCGACCAGGGGCA AGTAA 5 ' GATCGCAAGAGCACACGTCTGAACTCCACTCACGAGCATTGATCTCGTATGCCGTCTTCTGC TCTAGCCTT TCGCAGCACATCv C TTCTCACATv AGAGCGACCAGGGGCA AGTAA 5 '
DNA PCR-Free index 100 接头 DNA PCR-Free index 100 connector
The most stable dim r overall： 丄 2 bp, —22.8 kcsl/ o丄  The most stable dim r overall: 丄 2 bp, —22.8 kcsl/ o丄
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGCCGTGA CTCGTATGCCGTCTTCTGCTTG  5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGCCGTGA CTCGTATGCCGTCTTCTGCTTG
DNA PCR-Free index 101 接头 DNA PCR-Free index 101 connector
The most st ble cl Ime r overall： 12 bp, -22.8 kcal /mol  The most st ble cl Ime r overall: 12 bp, -22.8 kcal /mol
DNA PCR-Free index 102 接头 DNA PCR-Free index 102 connector
The most stable dimer overall： I 2 bp, -22.8 kcal mol  The most stable dimer overall: I 2 bp, -22.8 kcal mol
5'  5'
31 TCTAGCC CTCGCAG 3 1 TCTAGCC CTCGCAG
DNA PCR-Free index 103 接头  DNA PCR-Free index 103 connector
The most .st ble dirrier overall： 12 bp, -22■· S kcal ir-ol  The most .st ble dirrier overall: 12 bp, -22■· S kcal ir-ol
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTC— ¾CGGAATGGCATCT'CGTA CGTC^^^  5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTC — 3⁄4CGGAATGGCATCT'CGTA CGTC^^^
DNA PCR-Free index 104 接头 DNA PCR-Free index 104 connector
The mos stable diner overall: 丄 2 hpf -22.8 kcsl/ino丄 GGGCA AGTAA 5 'The mos stable diner overall: 丄2 hp f -22.8 kcsl/ino丄GGGCA AGTAA 5 '
DNA PCR-Free index 105 接头 DNA PCR-Free index 105 connector
The most stable diniei: ov rall： 12 bp, -22 , B kcal ir:ol  The most stable diniei: ov rall: 12 bp, -22 , B kcal ir:ol
5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCACGTCA ACATCTCGTATGCCGTCTTCTGCTTG  5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCACGTCA ACATCTCGTATGCCGTCTTCTGCTTG
A AGTAA A AGTAA
DNA PCR-Free index 106 接头DNA PCR-Free index 106 connector
he mo t s able diraer overall： 12 fo , -22. Θ kcal /mol  He mo t s able diraer overall: 12 fo , -22. Θ kcal /mol
5'  5'
3 ' TCTAGCCTTCTCGCAG  3 ' TCTAGCCTTCTCGCAG
DNA PCR-Free index 107 接头 ODNA PCR-Free index 107 connector O
?NCeede DAPRFrinxll7- ?NCeede DAPRFrinxll7-
Figure imgf000028_0001
Figure imgf000028_0001
p/he stable dimer overaJ b kcalmo- ?NCeede08 DAPRFr inxl- CTAGCCTIcrcGCAGCACAIT,c CTCACATCTAGAGCCACCAGCGGCATAGTAA:-. ...- Op/he stable dimer overaJ b kcalmo- ?NCeede08 DAPRFr inxl- CTAGCCTIcrcGCAGCACAIT,c CTCACATCTAGAGCCACCAGCGGCATAGTAA:-. ...- O
?NCeede DA PRFr inxl27- TCTA.GCC CTCGCAGCACATCCCTTTCTCACAl:CTAGAGiccACCAGCGGCATA.GT.AA ... NCeede DA PRFr inxl27- TCTA.GCC CTCGCAGCACATCCCTTTCTCACAl:CTAGAGiccACCAGCGGCATA.GT.AA ...
GATCGGAAGAGCACACGl GAAC!AGICACIGAC¾GA.CAT。^CGTATGCCCTCTTCrGCrPG ---_., GATCGGAAGAGCACACGl GAAC!AGICACIGAC3⁄4GA.CAT. ^CGTATGCCCTCTTCrGCrPG ---_.,
/The stable dimer overall kcalmo . /The stable dimer overall kcalmo .
?NCeede6 DA PRFr inxl2- p,/ The most stahLe overall b kcaJ_mo.--- ?NCeede5 DA PRFr inxl2- ACACGTCT:AGTCACTGArA cr:GrAc Ώ C TG;;r.,,  NCeede6 DA PRFr inxl2- p, / The most stahLe overall b kcaJ_mo.--- ?NCeede5 DA PRFr inxl2- ACACGTCT:AGTCACTGArA cr:GrAc Ώ C TG;;r.,,
Figure imgf000029_0001
Figure imgf000029_0001
?NCeede8 DA PRFr inxl 1- . , The most stable diirser overall： 12 fa , -22.8 ? NCeede8 DA PRFr inxl 1- . , The most stable diirser overall: 12 fa , -22.8
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGTCGTATCTCGTATGCCGTCTTCTGCTTG  5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGTCGTATCTCGTATGCCGTCTTCTGCTTG
DNA PCR-Free index 128 接头 DNA PCR-Free index 128 connector
The most s abl dimer overall： 2 p, kcal/ ol  The most s abl dimer overall: 2 p, kcal/ ol
3 J CTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCA AGTAA 5 '
Figure imgf000030_0001
3 J CTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCA AGTAA 5 '
Figure imgf000030_0001
1 TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCA AGTAA 5， 1 TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCA AGTAA 5,
DNA PCR-Free indexl30 接头 DNA PCR-Free indexl30 connector
The mos stable dim r 'm'e ll： 12 b , -22. S kcal irtol  The mos stable dim r 'm'e ll: 12 b , -22. S kcal irtol
，  ,
DNA PCR-Free index 131 接头 DNA PCR-Free index 131 connector
The most s able dimer overall： bp, kcal ATAG AA 5 ' The most s able dimer overall: bp, kcal ATAG AA 5 '
DNA PCR-Free index 132 接头
Figure imgf000030_0002
DNA PCR-Free index 132 connector
Figure imgf000030_0002
DNA PCR-Free indexl33 接头 DNA PCR-Free indexl33 connector
The most s able dimer overall : 12 fop, -22.8
Figure imgf000030_0003
The most s able dimer overall : 12 fop, -22.8
Figure imgf000030_0003
31 TCTAGCC TC CGCAGCACATCCCTT CTCACATCTAGAGCCACCAGCGGCATAGTi 3 1 TCTAGCC TC CGCAGCACATCCCTT CTCACATCTAGAGCCACCAGCGGCATAGTi
DNA PCR-Free index 134 接头  DNA PCR-Free index 134 connector
The most .st ble dimer overall 12 bp, -22.8 kcal/inol  The most .st ble dimer overall 12 bp, -22.8 kcal/inol
5， GATCGG.AAGAGCACAC  5, GATCGG.AAGAGCACAC
3 f CTAGCCTTC CGCAvGC 3 f CTAGCCTTC CGCAvGC
DNA PCR-Free indexl35 接头  DNA PCR-Free indexl35 connector
31 TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCA AGTAA 5 '3 1 TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCA AGTAA 5 '
DNA PCR-Free indexl36 接头 DNA PCR-Free indexl36 connector
The most stable dimer overall： 丄 2 bp, -22. S kcal /mo 1  The most stable dimer overall: 丄 2 bp, -22. S kcal /mo 1
5 ' GATCGGAAGAGCACACG CI  5 ' GATCGGAAGAGCACACG CI
DNA PCR-Free index 137 接头 The most st ble dirtier ov r ll： 12 r -22.8 kcal/iuol DNA PCR-Free index 137 connector The most st ble dirtier ov r ll: 12 r -22.8 kcal/iuol
： GCTTG 3 f : GCTTG 3 f
3 ' CTAGCCTTCTCGCAGCACATCCCTTTC CACA GTAGAGC ACCAGCGGCATAG AA 5 ' 3 ' CTAGCCTTCTCGCAGCACATCCCTTTC CACA GTAGAGC ACCAGCGGCATAG AA 5 '
DNA PCR-Free indexl39 接头  DNA PCR-Free indexl39 connector
The mo 1 stable dime r over ll： 12 b f -22.8 kc l /mol The mo 1 stable dime r over ll: 12 b f -22.8 kc l /mol
31 TCTAGCCTTCTCGCAGCACA CCCTTTCTCACATC AGAGCCACCAGv GGCATAGTAA 5， 3 1 TCTAGCCTTCTCGCAGCACA CCCTTTCTCACATC AGAGCCACCAGv GGCATAGTAA 5,
DNA PCR-Free indexl40 接头  DNA PCR-Free indexl40 connector
The most stable dimer overall： 12 fo f -22.8 kcal rnol The most stable dimer overall: 12 fo f -22.8 kcal rnol
5， GA CGGAAGAGCACACGTCTG.AACTCCAGTCACTGTAACCAATCTCGTATGCCGTC TCTGCTTG 3  5, GA CGGAAGAGCACACGTCTG.AACTCCAGTCACTGTAACCAATCTCGTATGCCGTC TCTGCTTG 3
DNA PCR-Free indexl41 接头 DNA PCR-Free indexl41 connector
The m t stable dimer overall： 12 bp, -22 , S kcal irtol  The m t stable dimer overall: 12 bp, -22 , S kcal irtol
； CTTG 3 ' ; CTTG 3 '
3 r TCTAGCCTTCTCGCAGCAGATCCCTTTCTCACATCTAGAGCCACCAGCC4GCATAGTAA 5 3 r TCTAGCCTTCTCGCAGCAGATCCCTTTCTCACATCTAGAGCCACCAGCC4GCATAGTAA 5
DNA PCR-Free indexl42 接头  DNA PCR-Free indexl42 connector
The mo t stable diraer overall： 丄 2 bpf -22.8 kcal /mo 1 The mo t stable diraer overall: 丄2 bp f -22.8 kcal /mo 1
3 ' 'TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3 ' 'TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free indexl43 接头  DNA PCR-Free indexl43 connector
The mo s t .stable diriier cveral 1： 12 bp, -22. S kc l /mol  The mo s t .stable diriier cveral 1: 12 bp, -22. S kc l /mol
5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC AACACCGATCTCGTATGCCGTCTTCTGCTTG 3  5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC AACACCGATCTCGTATGCCGTCTTCTGCTTG 3
DNA PCR-Free indexl44 接头 DNA PCR-Free indexl44 connector
The most stable dimer overall： 丄 2 pf - .8 kcal /mol The most stable dimer overall: 丄2 p f - .8 kcal /mol
5 ' GATCGGAAGi  5 ' GATCGGAAGi
DNA PCR-Free indexl45 接头 DNA PCR-Free indexl45 connector
The most .s able dimer overall： 12 bp f -22. S kcal TOO 1 The most .s able dimer overall: 12 bp f -22. S kcal TOO 1
3 f TCTAGCCTTCTGGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 51 3 f TCTAGCCTTCTGGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 1
DNA PCR-Free indexl46 接头  DNA PCR-Free indexl46 connector
The most stable dimer over ll： 12 b , -22.8 kcal/mo  The most stable dimer over ll: 12 b , -22.8 kcal/mo
3 f TCTAGCCTTCTCGCAGCAGATCCCT CTCACATC AGAGCCACCAGC GCA AGT^_ 5 ' 3 f TCTAGCCTTCTCGCAGCAGATCCCT CTCACATC AGAGCCACCAGC GCA AGT^_ 5 '
DNA PCR-Free indexl47 接头 The ost stable dimer overall： 12 b , -22.8 kcal mol DNA PCR-Free indexl47 connector The ost stable dimer overall: 12 b , -22.8 kcal mol
5 ' GATCGGAAGAGCACAG  5 ' GATCGGAAGAGCACAG
DNA PCR-Free index 148 接头 DNA PCR-Free index 148 connector
The most stable dir er overall： 12 bp, -22.8 kcsl/iriGl  The most stable dir er overall: 12 bp, -22.8 kcsl/iriGl
5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCAC GCC ATAATCTCGTATGCCGTCTTCTGCTTG  5 ' GA CGGAAGAGCACACGTCTGAACTCCAGTCAC GCC ATAATCTCGTATGCCGTCTTCTGCTTG
Figure imgf000032_0001
Figure imgf000032_0001
Figure imgf000032_0002
Figure imgf000032_0002
T TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 T T TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 T
DNA PCR-Free index 151 接头  DNA PCR-Free index 151 connector
The most stable dimer overal I： 丄 2 bp, -22.8 kcal/mol  The most stable dimer overal I: 丄 2 bp, -22.8 kcal/mol
5 f GATCGGAAGAGGACACCTCTG AC CCAG CAC GGATTAGATCTCGTATGCCGTCTTCTGCT G 35 f GATCGGAAGAGGACACCTCTG AC CCAG CAC GGATTAGATCTCGTATGCCGTCTTCTGCT G 3
31 CTAGCCTTCTCGCJ 3 1 CTAGCCTTCTCGCJ
DNA PCR-Free index 152 接头  DNA PCR-Free index 152 connector
The o st st ab丄 e dime r ove rail: -22.8 kcal/mol  The o st st ab丄 e dime r ove rail: -22.8 kcal/mol
5 ' GA CGGAAG2  5 ' GA CGGAAG2
3 ' TCTAGCC CTCGCAGt  3 ' TCTAGCC CTCGCAGt
DNA PCR-Free indexl53 接头  DNA PCR-Free indexl53 connector
The most stable diirier overall： 12 fop, -22. δ kcs丄 / ol  The most stable diirier overall: 12 fop, -22. δ kcs丄 / ol
5 ( GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACTGAGGATCTCGTATGCCGTCTTCTGC TG 3 5 ( GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACTGAGGATCTCGTATGCCGTCTTCTGC TG 3
DNA PCR-Free index 154 接头 DNA PCR-Free index 154 connector
The most stable diiner overall: 12 bp, -22.8 kcal /mo i  The most stable diiner overall: 12 bp, -22.8 kcal /mo i
Figure imgf000032_0003
Figure imgf000032_0003
DNA PCR-Free index 156 接头  DNA PCR-Free index 156 connector
The most s able d i jr- r overall： 12 b , -22 , S kcal /iitol  The most s able d i jr- r overall: 12 b , -22 , S kcal /iitol
DNA PCR-Free index 157 接头 The most stable dirr.er overall： 12 bp, -22 . 8 kcal mol DNA PCR-Free index 157 Connector The most stable dirr.er overall: 12 bp, -22 . 8 kcal mol
5 ' GATCGGAAGAGCACACGTCTGJL¾CTCCaGTCACTGGAAT CATCTCGTATGCCGTCTTCTGCTTG 3，  5 ' GATCGGAAGAGCACACGTCTGJL3⁄4CTCCaGTCACTGGAAT CATCTCGTATGCCGTCTTCTGCTTG 3,
DNA PCR-Free index 158 接头 DNA PCR-Free index 158 connector
The mo t stable dimer overall : 12 bp , -22 . 8 kcal /r o  The mo t stable dimer overall : 12 bp , -22 . 8 kcal /r o
ICT G 3，  ICT G 3,
3， TCTAGCCTTCTCGCAG  3, TCTAGCCTTCTCGCAG
DNA PCR-Free indexl59 接头  DNA PCR-Free indexl59 connector
mo t stable dimer overall： I 2 -22 . 6 kcal/mo丄  Mo t stable dimer overall: I 2 -22 . 6 kcal/mo丄
DNA PCR-Free index 160 接头DNA PCR-Free index 160 connector
h most stable dlmer oversl丄： 12 b 7 -22 . 8 kcal/mo上 h most stable dlmer oversl丄: 12 b 7 -22 . 8 kcal/mo
5 ' GATCGGAAGAGCACACG CTGAACTCCAGTCACAAGCGATTATCTCGTATGCCGTC TCTGCTTG 3  5 ' GATCGGAAGAGCACACG CTGAACTCCAGTCACAAGCGATTATCTCGTATGCCGTC TCTGCTTG 3
3 ? CTAGCCTTC CGCAGCAGATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 ' 3 ? CTAGCCTTC CGCAGCAGATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5 '
DNA PCR-Free index 161 接头  DNA PCR-Free index 161 connector
The most s able d inter overall： 12 bp -22 . 8 k.cal /iit.o.1  The most s able d inter overall: 12 bp -22 . 8 k.cal /iit.o.1
CCAGCGGCA AGTAA 5 ' CCAGCGGCA AGTAA 5 '
根据本发明的一些实施例， 本发明提供了一些 DNA PCR-Free标签接头， 这些 DNA According to some embodiments of the invention, the invention provides DNA PCR-Free tag junctions, these DNAs
PCR-Free标签接头由 DNA PCR-Free接头 1.0和 PCR-Free标签序列组成， 而这些 PCR-Free 标签序列包括如下或由如下组成： 表 1所示 161个 PCR-Free标签序列或与其所包含的 DNA标签序列相差 1个碱基的 PCR-Free标签序列中的至少 10个， 或至少 20个， 或至少 30 个， 或至少 40个， 至少 50个， 或至少 60个， 或至少 70个， 或至少 80个， 或 90个， 或至少 100个， 或至少 110个， 或至少 120个， 或至少 130个， 或至少 140个， 或至少 150个， 或全 部 161个。根据本发明的具体示例，这些 PCR-Free标签序列优选地至少包括表 1所示的 161 个 PCR-Free标签序列 中 的 PCR-Free Index- 1 ~ PCR-Free Index- 10 , 或 PCR-Free Index- 11 ~ PCR-Free Index-20, 或 PCR-Free Index-21 ~ PCR-Free Index-30 , 或 PCR-Free Index-31 ~ PCR-Free Index-40, 或 PCR-Free Index-41 ~ PCR-Free Index-50 , 或 PCR-Free Index-51 ~ PCR-Free Index-60, 或 PCR-Free Index-61 ~ PCR-Free Index-70 , 或 PCR-Free Index-71 ~ PCR-Free Index-80, 或 PCR-Free Index- 81 ~ PCR-Free Index-90 , 或 PCR-Free Index-91 - PCR-Free Index- 100 , 或 PCR-Free Index- 101 - PCR-Free Index- 110 , 或 PCR-Free Index-I l l ~ PCR-Free Index- 120 ,或 PCR-Free Index- 121 ~ PCR-Free Index- 130, 或 PCR-Free Index- 131 ~ PCR-Free Index- 140 , 或 PCR-Free Index- 141 ~ PCR-Free Index- 150, 或 PCR-Free Index- 151 ~ PCR-Free Index- 161 , 或者他们任何两个或多个的组 合。 根据具体的示例， 相差 1个碱基包括对标签序列中 1个碱基的取代、 添加或删除。 根 据本发明的实施例， 还提供了 DNA PCR-Free标签接头用于 DNA标签文库构建并测序的 用途。 由此， 根据本发明的实施例， 还提供了使用上述 DNA PCR-Free标签接头构建的 DNA标签文库。 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. According to a specific example of the present invention, 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-121 ~ PCR-Free Index-130, or PCR-Free Index-131 ~ PCR-Free Index-140, or PCR-Free Index- 141 ~ PCR -Free Index- 150, or PCR-Free Index- 151 ~ PCR-Free Index- 161, or a combination of any two or more of them. According to a specific example, a difference of 1 base includes substitution, addition or deletion of 1 base in the tag sequence. According to an embodiment of the present invention, a DNA PCR-Free tag linker is also provided for use in DNA tag library construction and sequencing. Thus, according to an embodiment of the present invention, a DNA tag library constructed using the above DNA PCR-Free tag linker is also provided.
根据本发明的另一方面， 本发明还提供了一种利用上述寡核苷酸 （DNA PCR-Free 标签接头）构建 DNA标签文库的方法。 具体地， 根据本发明的实施例， 参考图 2, 该方 法包括：  According to another aspect of the present invention, 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:
首先， 提供 DNA模板。 根据本发明的实施例， 该 DNA模板具有两条寡核苷酸链。 根据本发明的实施例， DNA样品的来源并不受特别限制， 可以来源于所有真核和原核 生物。 根据本发明的一个实施例， DNA样品为人 DNA样品， 更具体的， 可以为人基因 组 DNA样品。 根据本发明的实施例， 优选地， DNA模板的长度为约 250bp, 由此能够 进一步提高构建 DNA标签文库以及后续测序的效率。 发明人发现， 利用根据本发明实 施例的方法， 能够有效地构建多种常见模式生物的 DNA标签文库。 First, a DNA template is provided. According to an embodiment of the invention, the DNA template has two oligonucleotide strands. According to an embodiment of the present invention, the source of the DNA sample is not particularly limited and may be derived from all eukaryotic and prokaryotic Creature. According to one embodiment of the invention, the DNA sample is a human DNA sample, and more specifically, may be a human genomic DNA sample. According to an embodiment of the present invention, preferably, 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.
接下来， 在 DNA模板的两条寡核苷酸链的 3，末端分别添加碱基 A。 由此， 便获得 具有粘性末端 A的 DNA模板。 根据本发明的实施例， 在添加碱基 A前， 要先对 DNA 模板进行末端修复。  Next, base A is added to the 3's ends of the two oligonucleotide strands of the DNA template. Thus, a DNA template having a sticky end A was obtained. According to an embodiment of the present invention, the DNA template is subjected to end repair before the base A is added.
然后，在具有粘性末端 A的 DNA模板的两端分别连接含有选自上述根据本发明实施 例的一组分离的 DNA标签的一种的接头， 以便获得连接产物。 根据本发明的实施例， 该接头为选自根据本发明的实施例的一组分离的寡核苷酸的一种。 根据本发明的实施 例， 具有粘性末端 A的 DNA模板与 DNA PCR-Free标签接头， 是通过在具有粘性末端 A 的 DNA模板的两条寡核苷酸链的 3'末端均连接 DNA PCR-Free标签接头实现的。 上述根 据本发明的实施例所得到的 "连接产物" ， 含有目的片段、 DNA接头， 以及 DNA标签。 这里所使用的术语 "目的片段" ， 其序列与 DNA模板的序列相对应。 在这里， 目的片 段的序列与 DNA模板的序列相对应， 其含义是指， 可以通过目的片段的序列直接推导 出 DNA模板的序列， 例如， 目的片段的序列可以与 DNA模板的序列完全相同， 也可以 是完全互补， 甚至是增加或者减少了已知数目的已知碱基， 只要能够通过有限的计算获 得的 DNA的序列即可。  Then, 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. According to an embodiment of the invention, the linker is one selected from the group of isolated oligonucleotides according to embodiments of the invention. According to an embodiment of the present 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. The term "fragment of interest" as used herein, the sequence of which corresponds to the sequence of the DNA template. Here, the 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.
最后， 分离回收获得的连接产物， 这些连接产物构成 DNA 标签文库。 根据本发 明的实施例， 分离回收连接产物的方法不受特别限制， 本领域技术人员可以根据连接 产物的特点选择适当的方法和设备进行分离。 根据本发明的一个具体示例， 可以利用 2%的琼脂糖凝胶电泳分离回收所述连接产物。  Finally, the ligation products obtained are isolated and recovered, and these ligation products constitute a DNA tag library. According to the embodiment of the present invention, 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. According to a specific example of the present invention, the ligation product can be separated and recovered by 2% agarose gel electrophoresis.
进一步， 根据本发明的实施例， 本发明提供了一种构建 DNA标签文库的方法， 其 包括：  Further, in accordance with an embodiment of the present invention, the present invention provides a method of constructing a DNA tag library, comprising:
1 ) DNA 模板准备， 提供 n个 DNA样品， n为整数且 1 < n < 161的整数， 优选地 n为整数且 2 < n < 161 , 该 DNA样品可以来自所有真核和原核生物， 包括但不限于人 DNA样品； 优选地， 根据本发明的实施例， DNA模板长度为 250bp;  1) 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;
2 ) 末端修复；  2) end repair;
3 ) DNA模板 3，末端加 "A" 碱基；  3) DNA template 3, add "A" base at the end;
4 )连接 DNA标签接头， 以获得连接产物。 其中， 对不同 DNA模板使用不同的标签 接头， 每一个标签接头连接到 DNA模板的两端。 DNA标签接头， 是选自前面所述根据 本发明实施例的 DNA PCR-Free标签接头。  4) Connect the DNA tag linker to obtain the ligation product. Among them, different tag connectors are used for different DNA templates, and each tag connector is connected to both ends of the DNA template. The DNA tag linker is selected from the DNA PCR-Free tag linker described above according to an embodiment of the present invention.
5 ) 分离回收获得的连接产物， 这些连接产物构成 DNA 标签文库。 其中， 根据本 发明的实施例， 可以利用 2%的琼脂糖凝胶电泳分离回收连接产物。  5) The ligation products obtained are isolated and recovered, and these ligation products constitute a DNA tag library. Among them, according to an embodiment of the present invention, the ligation product can be separated and recovered by 2% agarose gel electrophoresis.
根据本发明的实施例， 通过上述根据本发明实施例的构建 DNA标签文库的方法所 构建的 DNA标签文库， 其 DNA PCR-Free标签接头由 DNA PCR-Free接头 1.0和 PCR-Free 标签序列组成， 这些 PCR-Free标签序列包括如下或由如下组成： 表 1所示 161个 PCR-Free 标签序列或与其所包含的 DNA标签序列相差 1个碱基的 PCR-Free标签序列中的至少 10 个， 或至少 20个， 或至少 30个， 或至少 40个， 至少 50个， 或至少 60个， 或至少 70个， 或 至少 80个， 或 90个， 或至少 100个， 或至少 110个， 或至少 120个， 或至少 130个， 或至少 140个， 或至少 150个， 或全部 161个。 上述根据本发明实施例的构建 DNA标签文库的方 法中，釆用的 PCR-Free标签序列优选地至少包括表 1所示的 161个 PCR-Free标签序列中的 PCR-Free Index- 1 ~ PCR-Free Index- 10 , 或 PCR-Free Index- 11 ~ PCR-Free Index-20 , 或 PCR-Free Index-21 ~ PCR-Free Index-30 , 或 PCR-Free Index-31 ~ PCR-Free Index-40 , 或 PCR-Free Index-41 ~ PCR-Free Index-50 , 或 PCR-Free Index-51 ~ PCR-Free Index-60 , 或 PCR-Free Index-61 ~ PCR-Free Index-70 , 或 PCR-Free Index-71 ~ PCR-Free Index-80 , 或 PCR-Free Index- 81 ~ PCR-Free Index-90, 或 PCR-Free Index-91 ~ PCR-Free Index- 100, 或 PCR-Free Index- 101 ~ PCR-Free Index- 110 ,或 PCR-Free Index-I l l ~ PCR-Free Index- 120, 或 PCR-Free Index- 121 ~ PCR-Free Index- 130 , 或 PCR-Free Index- 131 ~ PCR-Free Index- 140 , 或 PCR-Free Index- 141 - PCR-Free Index- 150 , 或 PCR-Free Index- 151 - PCR-Free Index-161 , 或者他们任何两个或多个的组合。 根据本发明的实施例， 相差 1 个碱基包括标签中 1个碱基的取代、 添加或删除。 According to an embodiment of the present invention, a DNA tag library constructed by the above method for constructing a DNA tag library according to an embodiment of the present invention, the DNA PCR-Free tag linker is composed of a DNA PCR-Free linker 1.0 and a PCR-Free tag sequence. These PCR-Free tag sequences include or consist of: 161 PCR-Free tag sequences shown in Table 1 or at least 10 of the PCR-Free tag sequences differing by one base from the DNA tag sequence contained therein, 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. In the above method for constructing a DNA tag library according to an embodiment of the present invention, the PCR-Free tag sequence preferably comprises at least PCR-Free Index-1 to PCR- in the 161 PCR-Free tag sequences shown in Table 1. Free Index- 10 , 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-121 ~ PCR-Free Index-130, or PCR-Free Index-131 ~ PCR-Free Index-140, or PCR-Free Index- 141 - PCR- Free Index-150, or PCR-Free Index-151 - PCR-Free Index-161, or a combination of any two or more of them. According to an embodiment of the invention, a difference of 1 base comprises a substitution, addition or deletion of 1 base in the tag.
利用根据本发明实施例的构建 DNA标签文库的方法， 能够有效地将根据本发明实 施例的 DNA标签引入到针对 DNA样品所构建的 DNA标签文库中。 从而可以通过对 DNA标签文库进行测序， 获得 DNA样品的序列信息以及 DNA标签的序列信息， 从而 能够对 DNA样品的来源进行区分。 另外， 发明人惊奇地发现， 当针对相同的样品， 基 于上述方法， 釆用具有不同标签的寡核苷酸构建含有各种 DNA标签的 DNA标签文库 时， 所得到的测序数据结果的稳定性和可重复性非常好。  With the method of constructing a DNA tag library according to an embodiment of the present invention, a DNA tag according to an embodiment of the present invention can be efficiently introduced into a DNA tag library constructed for a DNA sample. Thus, by sequencing the DNA tag library, the sequence information of the DNA sample and the sequence information of the DNA tag can be obtained, thereby distinguishing the source of the DNA sample. In addition, 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.
根据本发明的实施例， 本发明对 Illumina提供的 DN A接头序列进行优化， 在接头 中引入标签序列， 通过 DNA PCR-Free标签接头的连接将标签序列导入目的文库中。 在 接头连接后， 只需分离回收连接产物即可得到目的文库， 整个建库过程无需经过 PCR 反应， 避免了由于 PCR反应的难度大， 特异性低而造成 DNA标签文库构建效率低下的 问题， 同时也降低了文库构建的费用。 目前为止， 通过这些 DNA PCR-Free标签接头导 入标签的 DNA文库构建方法及其标签序列，并没有相关的报道。根据本发明的实施例， 与 Illumina公司的 DNA接头相比， 本发明的 DNA标签接头， 是优化后的 DNA标签接 头， 这些 DNA标签接头提高了接头连接的效率， 并提高了标签序列的识别效率及标签 的数量。 具体情况， 可比较参照图 1和图 2, 其中图 1所示的 Illumina公司的 DNA 标 签文库构建方法的流程图，图 2所示的根据本发明的实施例的 DNA 标签文库构建方法 的流程图。  According to an embodiment of the present invention, the present invention optimizes the DN A linker sequence provided by Illumina, introduces a tag sequence into the adaptor, and introduces the tag sequence into the library of interest by ligation of a DNA PCR-Free tag linker. After the linker is connected, the target library can be obtained by separately separating and recovering the ligation product, and the whole process of building the library does not need to undergo a PCR reaction, thereby avoiding the problem that the efficiency of the PCR reaction is low and the specificity is low, thereby preventing the construction of the DNA tag library from being inefficient. It also reduces the cost of library construction. So far, the DNA library construction methods and their tag sequences introduced into these tags by these DNA PCR-Free tag linkers have not been reported. According to an embodiment of the present invention, the DNA tag linker of the present invention is an optimized DNA tag linker as compared with the DNA linker of Illumina Corporation, and these DNA tag linkers improve the efficiency of the linker connection and improve the recognition efficiency of the tag sequence. And the number of labels. Specifically, a comparison can be made with reference to FIG. 1 and FIG. 2, wherein a flowchart of a method for constructing a DNA tag library of Illumina Corporation shown in FIG. 1 and a flowchart of a method for constructing a DNA tag library according to an embodiment of the present invention shown in FIG. .
根据本发明的再一方面， 本发明还提供了一种用于构建 DN A标签文库的试剂盒。 根据本发明的实施例， 该试剂盒包括： 161种分离的寡核苷酸， 这些分离的寡核苷酸具 有第一链和第二链， 其中， 第一链由 SEQ ID NO: 323所示的核苷酸构成， 第二链由 SEQ ID NO: ( 2N ) 所示的核苷酸构成， 其中 N=l-161的整数。 其中， 这 161种分离 的寡核苷酸分别设置在不同的容器中。 由此， 利用该试剂盒， 能够方便地将根据本发明 实施例的 DNA标签引入到构建的 DNA标签文库中。 当然， 本领域技术人员能够理解， 试剂盒中还可以包含其他用于构建 DNA标签文库的常规组件， 在此不再赘述。  According to still another aspect of the present invention, the present invention also provides a kit for constructing a DN A tag library. According to an embodiment of the invention, the kit comprises: 161 isolated oligonucleotides having a first strand and a second strand, wherein the first strand is represented by SEQ ID NO: 323 The nucleotide structure consists of the nucleotide represented by SEQ ID NO: (2N), wherein N = an integer of from 1 to 161. Among them, the 161 isolated oligonucleotides were set in different containers. Thus, with the kit, a DNA tag according to an embodiment of the present invention can be conveniently introduced into a constructed DNA tag library. Of course, those skilled in the art can understand that other components for constructing a DNA tag library can also be included in the kit, and details are not described herein.
DNA标签文库及测序方法  DNA tag library and sequencing method
根据本发明的又一方面， 本发明还提供了一种 DNA标签文库， 其是根据本发明的 构建 DNA标签文库的方法所构建的。 该具有标签的 DNA标签文库可以有效地应用于 高通量测序技术例如 Solexa技术， 从而可以通过获得标签序列， 来对所获得的核酸序 列信息例如 DNA序列信息来精确地进行样品来源分类。  According to still another aspect of the present invention, the present invention also provides a DNA tag library constructed according to the method of constructing a DNA tag library of the present invention. The tagged DNA tag library can be effectively applied to high-throughput sequencing technologies such as Solexa technology, so that the obtained nucleic acid sequence information such as DNA sequence information can be accurately classified by sample source by obtaining a tag sequence.
根据本发明的又一方面， 本发明还提供了一种确定 DNA样品序列信息的方法。 根 据本发明的实施例， 其包括： 根据本发明实施例的构建 DNA 标签文库的方法， 构建 DNA标签文库； 接着， 对所构建的 DNA标签文库进行测序， 以确定 DNA样品的序列 信息。 基于该方法， 能够有效地获得 DNA标签文库中 DNA样品的序列信息以及 DNA 标签的序列信息， 从而能够对 DNA样品的来源进行区分。 另外， 发明人惊奇地发现， 利用根据本发明实施例的方法确定 DNA样品序列信息， 能够有效地减少数据产出偏向 性的问题， 并且能够精确地对多种 DNA标签文库进行区分。 根据本发明的实施例， 可 以釆用任何已知的方法对所构建的 DNA标签文库进行测序， 其类型并不受特别限制。 根据本发明的一些示例， 可以利用 Solexa测序技术对 DNA标签文库进行测序。 根据本 发明的实施例， 可以根据具体情况选择合适的测序引物进行测序。  According to still another aspect of the present invention, the present invention also provides a method of determining DNA sample sequence information. According to an embodiment of the present invention, the method comprises: constructing a DNA tag library according to a method for constructing a DNA tag library according to an embodiment of the present invention; and then, sequencing the constructed DNA tag library to determine sequence information of the DNA sample. Based on this method, 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 distinguishing the source of the DNA sample. Further, 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 output bias, and can accurately distinguish a plurality of DNA tag libraries. According to an embodiment of the present invention, the constructed DNA tag library can be sequenced by any known method, and the type thereof is not particularly limited. According to some examples of the invention, DNA tag libraries can be sequenced using Solexa sequencing technology. According to an embodiment of the present invention, suitable sequencing primers can be selected for sequencing according to specific conditions.
进一步， 可以将上面确定 DNA样品序列信息的方法应用于多种样品。 例如， 根据 本发明的实施例， 本发明提供了一种确定多种 DNA样品序列信息的方法。 根据本发明 的实施例， 其包括以下步骤： 针对多种样品的每一种， 分别独立地根据根据本发明的实 施例的构建 DNA标签文库的方法， 构建该 DNA样品的 DNA标签文库， 其中， 不同的 DNA样品釆用相互不同并且已知序列的 DNA标签，这里所使用的术语"多种"为 2-161 种。 将得到的多种样品的 DNA标签文库进行组合， 以便获得 DNA标签文库混合物。 利用 Solexa测序技术， 对所得的 DNA标签文库混合物进行测序， 从而获得 DNA样品 的序列信息以及标签的序列信息。 最后， 基于标签的序列信息， 对 DNA样品的序列信 息进行分类， 以便确定所述多种 DNA样品的序列信息。 由此， 根据本发明实施例的该 方法， 可以充分利用高通量的测序技术， 例如利用 Solexa测序技术， 同时对多种样品 的 DNA文库进行测序， 从而提高 DNA文库测序的效率和通量， 同时可以提高确定多 种 DNA样品的序列信息的效率。 关于测序的方法和釆用的测序引物， 前面已经进行了 详细描述， 此处不再赞述。 Further, the method of determining the DNA sample sequence information above can be applied to a plurality of samples. For example, according to In an embodiment of the invention, the invention provides a method of determining sequence information for a plurality of DNA samples. According to an embodiment of the present invention, the method comprises the steps of: constructing a DNA tag library of the DNA sample according to a method for constructing a DNA tag library according to an embodiment of the present invention, respectively, for each of a plurality of samples, wherein Different DNA samples use DNA labels of different and known sequences, and the term "various" is used herein to be 2-161. The resulting DNA tag libraries of various samples were combined to obtain a DNA tag library mixture. The resulting DNA tag library mixture is sequenced using Solexa sequencing technology to obtain sequence information of the DNA sample and sequence information of the tag. Finally, based on the sequence information of the tag, the sequence information of the DNA sample is classified to determine sequence information of the plurality of DNA samples. Thus, 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 to simultaneously sequence DNA libraries of various samples, thereby improving the efficiency and throughput of DNA library sequencing. At the same time, the efficiency of determining sequence information of a plurality of DNA samples can be improved. The methods for sequencing and the sequencing primers used in the prior art have been described in detail above and will not be described here.
需要说明的是， 根据本发明实施例的确定 DNA样品序列信息的方法是本申请的发 明人经过艰苦的创造性劳动和优化工作才完成的。 下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解， 下面的实 施例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注明具体技术或条 件的， 按照本领域内的文献所描述的技术或条件（例如参考 J.萨姆布鲁克等著， 黄培堂 等译的 《分子克隆实验指南》 ， 第三版， 科学出版社）或者按照产品说明书进行。 所用 试剂或仪器未注明生产厂商者， 均为可以通过市购获得的常规产品， 例如可以釆购自 Illumina公司。 实施例 1  It should be noted that the method of determining the DNA sample sequence information according to an embodiment of the present invention is completed by the inventor of the present application through arduous creative labor and optimization work. The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. Where the specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer, and are commercially available products, such as those available from Illumina. Example 1
Paired End DNA寡核苷酸序列：
Figure imgf000036_0001
Paired End DNA Oligonucleotide Sequence:
Figure imgf000036_0001
GATCT GATCT
DNA PCR-Free index接头： (如表 1所示） 实施例 1: DNA PCR-Free index connector: (as shown in Table 1) Example 1:
1.1 DNA 模板准备  1.1 DNA template preparation
以质粒 pMD18-T ( 日本 takara )为模板， 使用 Primer Premier5.0软件设计引物， PCR 扩增产物长度为 250bp的片段， 使用 NanoDrop 1000仪器（美国 NanoDrop )检测扩增产物 的浓度， 然后根据浓度取 1微克的该 PCR产物作为文库构建的插入片段， 补水使其体积 至 35微升。 PCR引物序列：  The plasmid pMD18-T (Japanese takara) was used as a template, primers were designed using Primer Premier 5.0 software, and a fragment of 250 bp in length was amplified by PCR. The concentration of the amplified product was detected using a NanoDrop 1000 instrument (NanoDrop, USA), and then the concentration was determined according to the concentration. One microgram of this PCR product was used as an insert for library construction and hydrated to a volume of 35 microliters. PCR primer sequence:
pMD18-T引物 1:CGGGGAGAGGCGGTTTGCGTATTGG；  pMD18-T primer 1: CGGGGAGAGGCGGTTTGCGTATTGG;
pMD 18-T引物 2:TTTTGTGATGCTCGTCAGGGGGGCG。  pMD 18-T primer 2: TTTTGTGATGCTCGTCAGGGGGGCG.
1.2 末端修复  1.2 End repair
按照下列的配比准备反应混合：  Prepare the reaction mixture according to the following ratio:
pMD 18-T质粒 DNA模板 35 微升  pMD 18-T plasmid DNA template 35 μL
T4 DNA 连接酶緩冲液 50 微升  T4 DNA ligase buffer 50 μl
dNTPs 混合液 4 微升  dNTPs mixture 4 μL
T4 DNA聚合酶 5 微升 Klenow DNA聚合酶 1 微升 T4 DNA polymerase 5 μl Klenow DNA Polymerase 1 μL
T4多聚核苷酸激酶 5 微升  T4 polynucleotide kinase 5 μL
总体积 100 微升  Total volume 100 microliters
将舒适型恒温混匀器调至 20 °C , 反应 30min 然后用 QIAquick PCR纯化试剂盒进行 纯化， 最后将样品溶于 32 i升 EB solution。  The comfort thermomixer was adjusted to 20 °C, reacted for 30 min and then purified using the QIAquick PCR Purification Kit, and finally the sample was dissolved in 32 μl of EB solution.
1.3 DNA片段 3'末端力口 "A"碱基  1.3 DNA fragment 3' end force "A" base
按照下列的配比准备反应混合物：  Prepare the reaction mixture according to the following ratio:
末端修复后的 DNA 32 微升  32 μL of DNA after end repair
Klenow 酶緩冲液 5 微升  Klenow Enzyme Buffer 5 μL
dATP(lmM) 10 微升  dATP (lmM) 10 microliters
Klenow 酶 (3'到 5' 外切酶活性） 3 微升  Klenow enzyme (3' to 5' exonuclease activity) 3 microliters
总体积 50 微升  Total volume 50 μl
将舒适型恒温混匀器调至 37 °C , 反应 3 Omin， 然后用 MiniElute PCR纯化试剂盒进行 纯化， 最后将样品溶于 10微升 EB solution。  The comfort thermomixer was adjusted to 37 °C for 3 Omin, then purified using the MiniElute PCR Purification Kit and finally dissolved in 10 μl of EB solution.
1.4 连接 PCR-Free标签接头  1.4 Connection PCR-Free Tag Connector
按照下列的配比准备反应混合物：  Prepare the reaction mixture according to the following ratio:
DNA 10 微升  DNA 10 μl
T4 DNA 连接酶緩冲液 25 微升  T4 DNA ligase buffer 25 μl
DNA PCR-Free标签接头 10 微升  DNA PCR-Free Tag Connector 10 μL
T4DNA 连接酶 5 微升  T4DNA ligase 5 μL
总体积 50 微升  Total volume 50 μl
注： 对于每一个 DNA样品， 所使用的 PCR-Free标签接头可为表 1中所示 PCR-Free标 签序列 （ PCR-Free Index-N ) 中的任意一种与 DNA PCR-Free接头 1.0退火后的 PCR-Free 标签接头。  Note: For each DNA sample, the PCR-Free tag linker used can be annealed to the DNA PCR-Free Linker 1.0 after any of the PCR-Free tag sequences (PCR-Free Index-N) shown in Table 1. PCR-Free tag connector.
将舒适型恒温混匀器调至 20°C , 反应 15min, 然后用 QIAquick PCR纯化试剂盒进行 纯化， 最后将样品溶于 30微升 EB solution 0 The comfort thermostat mixer was adjusted to 20 ° C for 15 min, then purified using the QIAquick PCR purification kit, and finally the sample was dissolved in 30 μl EB solution 0
1.5 PCR-Free index文库的胶回收纯化  1.5 PCR-Free index library for gel recovery and purification
将连接产物于 2%的琼脂糖胶中进行电泳分离； 随后将目的片段条带切胶转移至 The ligation product was electrophoretically separated in 2% agarose gel; then the target fragment strip was transferred to the gelatin to
Eppendorf管中。 用 QIAquick胶纯化试剂盒进行胶纯化回收， 回收产物溶于 20微升 EB solution。 In the Eppendorf tube. The gel was purified by QIAquick gel purification kit, and the recovered product was dissolved in 20 μl of EB solution.
1.6 PCR-Free index文库检测  1.6 PCR-Free index library detection
1 )使用 Agilent 2100 Bioanalyzer检测文库产量。  1) Library yield was measured using an Agilent 2100 Bioanalyzer.
2 )使用 QPCR定量检测文库产量。  2) Quantitative detection of library yield using QPCR.
图 3显示了根据本实施例的构建的 44个 D N A标签文库的电泳结果。 将构建的 44个 DNA PCR-Free 标签文库， 使用 1%琼脂糖凝胶电泳， 结果如图 3所示。 其中， （a ) 中 箭头所标记的为目的文库， 其中泳道 1和泳道 25分别是 D2000和 50bp的 marker, 泳道 2-24 分别是 DNA PCR-Free标签 1文库至 DNA PCR-Free标签 23文库。 （b ) 中箭头所标记的为 目的文库， 其中泳道 1和泳道 25分别是 D2000和 50bp的 marker , 泳道 3-23分别是 DNA PCR-Free标签 101文库至 DNA PCR-Free标签 121文库。 由于目的片段 （PCR产物） 长度 为 250bp,接头长度为 65bp且为 "Y型"结构，理论上目的片段两端连接上 DNA PCR-Free 标签接头后的会增加 130bp左右的分子量， 但是由于 DNA PCR-Free标签接头为特殊的 "Y型" 结构， 其在琼脂糖中电泳的速度会比相同分子量的双链 DNA电泳迁移率稍小， 电泳条带显示的位置为 500bp。 其中， D2100 marker从上往下的条带分别是： 2000bp、 1000bp、 750bp、 500bp、 250bp、 lOObp; 目的条带下方有部分条带， 为目的片段的一端 连接上接头的产物， 其产物量不多， 不影响文库构建的结果。 结果表明， 接头全部成功 连接到 PCR产物上。 Figure 3 shows the results of electrophoresis of 44 DNA tag libraries constructed according to the present example. The 44 DNA PCR-Free tag libraries to be constructed were electrophoresed on a 1% agarose gel, and the results are shown in Fig. 3. Wherein, (a) the arrow indicated by the middle arrow is the target library, wherein lane 1 and lane 25 are D2000 and 50 bp markers, respectively, and lanes 2-24 are DNA PCR-Free tag 1 library to DNA PCR-Free tag 23 library, respectively. (b) The target library is labeled with the middle arrow, wherein lanes 1 and 25 are D2000 and 50 bp markers, respectively, and lanes 3-23 are DNA PCR-Free tag 101 library to DNA PCR-Free tag 121 library, respectively. Due to the length of the fragment of interest (PCR product) It is 250 bp, the linker is 65 bp in length and has a "Y-type" structure. Theoretically, the DNA fragment of the target fragment attached to the DNA PCR-Free tag link will increase the molecular weight of about 130 bp, but the DNA PCR-Free tag linker is special." The Y-type structure, which is electrophoresed in agarose, has a slightly lower electrophoretic mobility than the double-stranded DNA of the same molecular weight, and the electrophoretic band shows a position of 500 bp. Among them, the bands of D2100 marker from top to bottom are: 2000bp, 1000bp, 750bp, 500bp, 250bp, lOObp; there are some bands below the target band, which is the product of the linker at one end of the target fragment, and the product quantity is not More, does not affect the results of library construction. The results showed that all of the linkers were successfully linked to the PCR product.
Solexa测序结果统计： 共测序 33932756条读取结果（也称为 reads ) , 标签完全识别 即 0 mismatch占 96.73% , 其他读取结果（ other reads ) 占 3.27% , 所以测序结果标签的完 全识别的序列为〜 96.7% , 可以满足 solexa DNA 标签的测序要求。  Solexa sequencing results statistics: a total of 33,932,756 reads (also known as reads), the tag is fully recognized, ie 0 mismatch, accounting for 96.73%, and other reads (3.2%), so the fully identified sequence of the sequencing result tags For ~ 96.7%, the sequencing requirements for the Solexa DNA tag can be met.
工业实用性 Industrial applicability
本发明的用于构建 DNA标签文库的 DNA标签、 寡核苷酸、 DNA标签文库及其制 备方法、 确定 DNA样品序列信息的方法、 确定多种 DNA样品序列信息的方法以及用 于构建 DNA标签文库的试剂盒， 能够应用于 DNA测序， 并且能够有效地提高测序平 台， 例如 Solexa测序平台的测序通量。  A DNA tag, an oligonucleotide, a DNA tag library, a preparation method thereof, a method for determining DNA sample sequence information, a method for determining sequence information of a plurality of DNA samples, and a DNA tag library for constructing a DNA tag library of the present invention The kit can be applied to DNA sequencing and can effectively improve the sequencing throughput of sequencing platforms such as the Solexa sequencing platform.
尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根 据已经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的 保护范围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。  Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations may be made to those details, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
在本说明书的描述中， 参考术语 "一个实施例" 、 "一些实施例" 、 "示意性实施 例" 、 "示例" 、 "具体示例" 、 或 "一些示例" 等的描述意指结合该实施例或示例描 述的具体特征、 结构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说 明书中， 对上述术语的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具 体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结 合。  In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

权利要求书 Claim
I.一组分离的 DNA标签，其由 SEQ ID NO: ( 2N-1 )所示的核苷酸构成，其中 N=l-161 的任意整数。 A set of isolated DNA tags consisting of the nucleotides set forth in SEQ ID NO: (2N-1), wherein N = any integer from 1 to 161.
2、 一组分离的寡核苷酸， 所述分离的寡核苷酸具有第一链和第二链， 所述第一链 由 SEQ ID NO: 323所示的核苷酸构成， 所述第二链分别由 SEQ ID NO: ( 2N ) 所示的 核苷酸构成， 其中 N=l-161的任意整数。  2. A set of isolated oligonucleotides having a first strand and a second strand, said first strand being comprised of a nucleotide set forth in SEQ ID NO: 323, said The two strands are each composed of a nucleotide represented by SEQ ID NO: (2N), wherein N = any integer of 1-161.
3、 一种制备 DNA标签文库的方法， 其特征在于， 包括以下步骤：  3. A method of preparing a DNA tag library, comprising the steps of:
提供 DNA模板， 所述 DNA模板具有两条寡核苷酸链；  Providing a DNA template having two oligonucleotide strands;
在所述 DN A模板的两条寡核苷酸链的 3，末端分别添加碱基 A；  Adding base A at the 3' end of the two oligonucleotide strands of the DN A template;
在所述 DNA模板的两端分别连接含有选自权利要求 1所述的一组分离的 DNA标签 的一种的接头， 以便获得连接产物； 以及  A linker comprising one selected from the group consisting of the set of isolated DNA tags of claim 1 is ligated to both ends of the DNA template to obtain a ligation product;
分离回收所述连接产物， 所述连接产物构成所述 DNA标签文库。  The ligation product is isolated and isolated, and the ligation product constitutes the DNA tag library.
4、 根据权利要求 3所述的制备 DNA标签文库的方法， 其特征在于，  4. A method of preparing a DNA tag library according to claim 3, wherein
所述接头为选自根据权利要求 2所述的一组分离的寡核苷酸的一种。  The linker is one selected from the group of isolated oligonucleotides according to claim 2.
5、 根据权利要求 3所述的制备 DNA标签文库的方法， 其特征在于，  5. The method of preparing a DNA tag library according to claim 3, wherein
所述 DNA模板的长度为约 250bp。  The DNA template is about 250 bp in length.
6、 根据权利要求 3所述的制备 DNA标签文库的方法， 其特征在于，  6. The method of preparing a DNA tag library according to claim 3, wherein
利用 2%的琼脂糖凝胶电泳分离回收所述连接产物。  The ligation product was separated and recovered by 2% agarose gel electrophoresis.
7、 根据权利要求 3所述的制备 DNA标签文库的方法， 其特征在于，  7. The method of preparing a DNA tag library according to claim 3, wherein
所述 DNA模板来自于人 DNA样品。  The DNA template is derived from a human DNA sample.
8、 根据权利要求 3所述的制备 DNA标签文库的方法， 其特征在于，  8. The method of preparing a DNA tag library according to claim 3, wherein
在添加碱基 A之前， 进一步包括对所述 DNA模板进行末端修复的步骤。  Prior to the addition of base A, a step of end repairing the DNA template is further included.
9、 一种 DNA标签文库， 其是根据权利要求 3-8任一项所述的方法建立的。  9. A DNA tag library constructed according to the method of any of claims 3-8.
10、 一种确定 DNA样品序列信息的方法， 其包括以下步骤：  10. A method of determining DNA sample sequence information, comprising the steps of:
根据权利要求 3-8任一项所述的方法， 建立所述 DNA样品的 DNA标签文库； 以 及  The method according to any one of claims 3-8, wherein the DNA tag library of the DNA sample is established;
对所述 DNA标签文库进行测序， 以确定所述 DNA样品的序列信息。  The DNA tag library is sequenced to determine sequence information for the DNA sample.
I I、 根据权利要求 10所述的确定 DNA样品序列信息的方法， 其特征在于， 对所述 DNA标签文库进行测序是利用 Solexa测序技术进行的。  I I. The method for determining DNA sample sequence information according to claim 10, wherein the sequencing of the DNA tag library is performed by Solexa sequencing technology.
12、 一种确定多种 DNA样品序列信息的方法， 其包括下列步骤：  12. A method of determining sequence information for a plurality of DNA samples, the method comprising the steps of:
针对所述多种样品的每一种， 分别独立地根据权利要求 3-8任一项所述的方法， 建 立所述 DNA样品的 DNA标签文库， 其中， 不同的 DNA样品釆用相互不同并且已知序 列的 DNA标签， 其中所述多种为 2-161种；  For each of the plurality of samples, a DNA tag library of the DNA sample is independently established according to the method of any one of claims 3-8, wherein different DNA samples are different from each other and have been used Knowing the DNA tag of the sequence, wherein the plurality of types are 2-161;
将所述多种样品的 DNA标签文库进行组合， 以便获得 DNA标签文库混合物； 利用 Solexa测序技术， 对所述 DNA标签文库混合进行测序， 以获得所述 DNA样 品的序列信息以及所述标签的序列信息； 以及  Combining DNA library libraries of the plurality of samples to obtain a DNA tag library mixture; sequencing the DNA tag library by Solexa sequencing technology to obtain sequence information of the DNA sample and sequence of the tag Information;
基于所述标签的序列信息对所述 DNA样品的序列信息进行分类， 以便确定所述多 种样品的 DN A序列信息。  The sequence information of the DNA sample is classified based on the sequence information of the tag to determine DN A sequence information of the plurality of samples.
13、 一种用于构建 DNA标签文库的试剂盒， 其包括：  13. A kit for constructing a DNA tag library, comprising:
161种分离的寡核苷酸， 所述分离的寡核苷酸具有第一链和第二链， 所述第一链由 SEQ ID NO: 323所示的核苷酸构成， 所述第二链由 SEQ ID NO: ( 2N )所示的核苷酸 构成， 其中 N=l-161的整数，  161 isolated oligonucleotides having a first strand and a second strand, the first strand consisting of the nucleotide set forth in SEQ ID NO: 323, the second strand It is composed of the nucleotide represented by SEQ ID NO: (2N), wherein N = an integer of 1-161,
其中， 所述 161种分离的寡核苷酸分别设置在不同的容器中。  Wherein the 161 isolated oligonucleotides are respectively disposed in different containers.
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